Novel piperidine carboxamide compound, preparation method, and usage thereof

ABSTRACT

The present invention provides a compound of a general formula I, a stereomeride, pharmaceutically acceptable salt or a solvate thereof, a preparation method thereof, and usages in preparing medicines for preventing and/or treating diseases or symptoms caused by drug-resistant tumors or drug-resistant bacteria and in preparing medicines for preventing and/or treating diseases or symptoms related to tumors, neurodegenerative diseases, allogeneic transplantation rejection, and infection. Preferably, the diseases or symptoms related to tumors, neurodegenerative diseases, allogeneic transplantation rejection, and infection are diseases or symptoms caused by a heat shock protein 70 (Hsp70). The compound in the present invention is used for overcoming a difficult problem of drug resistance of tumors, improves the effect of tumor treatment, and provides a new medical strategy for clinical tumor treatment.

TECHNICAL FIELD

The present invention is in the field of medicine, and specificallyrelates to novel piperidine carboxamide compound, preparation method,and use thereof.

BACKGROUND ART

Heat shock protein 70 (Hsp70) is widely present in nucleus, cytoplasm,endoplasmic reticulum, mitochondria and chloroplast cells, and isinvolved in intracellular protein de novo synthesis, orientation,protein maturation and degradation of misfolded proteins, thus affectingthe growth and metabolism function of cells. In cells, Hsp70 bindingtonascent polypeptides on ribosome can prevent misfolding of nascentpolypeptides; Hsp70 is essential for remodeling clathrin in the processof pinocytosis of mammalian cells; and Hsp70 binding to non nativeconformation proteins can promote proper folding and assembly ofproteins, and can maintain extended conformation of protein precursorsand prevent their aggregation denaturation and degradation, allowingeasy transport to organelles.

Studies have shown that, Hsp70 is related to many diseases, such astumors, neurodegenerative diseases, allograft rejection or infection andthe like. In cancer cells, Hsp70 affects apoptosis mainly through thefollowing pathways: (1) mitochondrial pathway: in the early stage ofmitochondria, Hsp70 blocks migration of Bax, and decreases permeabilityof mitochondrial outer membrane, thereby inhibiting the release of cytcand AIF from mitochondria; in the late stage of mitochondria, Hsp70binds directly to Apaf1, blocks the aggregation of procaspase-9, so thatapoptotic body cannot be formed, and caspase-3 downstream cannot beactivated; (2) death receptor pathway: Hsp70, by inhibiting theactivation of JNK, and binding to Akt and PKC, triggersdephosphorylation of kinase, and allows protein stabilization, cellsurvival; similarly, Hsp70 can also bind to DR4 and DR5, and inhibitTRAIL-induced DISC aggregation and activity; (3) DNA degradationpathway: the complex of Hsp70, Hsp40, ICAD can inhibit the activity andfolding effect of DNase CAD, prevent late apoptotic chromosomal DNA frombeing degraded, so as to achieve anti-apoptosis effect.

Study on Hsp70 useful for tumor therapy has become a hot spot in recentyears, but a highly active inhibitor has not found yet, and themechanism of action is not clear. In tumor cells, Hsp70 and its relatedprotein expression are abnormally high. Experiments prove that afterdosing stimulation tumor cells play a potential defense mechanism viaprotein Hsp70 to produce drug resistance, causing a decrease in activityof the drug. Hsp70 inhibitor is expected to reverse the antitumor drugresistance of tumor cell lines.

Contents of the Invention

The inventors of the present invention design and synthesize a type ofHsp70 inhibitors having novel structure, which can be used for theprevention or treatment of Hsp70-related diseases or conditions, and caneffectively reverse drug resistance of bacteria or tumor cells. Thepresent invention is accomplished just based on the above discovery.

In the first aspect, the present invention provides a compound offormula (I), an isomer, a pharmaceutically acceptable salt or a solvatethereof,

wherein:

A represents CH₂, S, O,

wherein “

” terminal is attached to R₂;

R₁ represents aryl, aromatic heterocyclyl, arylalkyl, or aromaticheterocyclylalkyl, wherein said aryl, aromatic heterocyclyl, arylalkyl,or aromatic heterocyclylalkyl is unsubstituted or substituted with oneor two substituents independently selected from the group consisting ofhalogen, nitro, hydroxy, amino, cyano, alkyl, cycloalkyl, alkoxy,alkylthio, alkylamino, cycloalkoxy, cycloalkylthio, cycloalkylamino,alkenyl and alkynyl;

R₂ represents hydrogen, alkyl, cycloalkyl, substituted cycloalkyl,alkoxy, alkoxycarbonyl, alkanoyl, substituted alkanoyl, aliphaticheterocyclyl, substituted aliphatic heterocyclyl, aliphaticheterocyclylalkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, arylsulfonyl, substituted arylsulfonyl, arolyl, substitutedarolyl, aromatic heterocyclyl, substituted aromatic heterocyclyl,aromatic heterocyclylalkyl, aromatic heterocyclylsulfonyl, aromaticheterocyclylacyl; wherein the substituent includes C₁-C₆alkyl, halogen,nitro, cyano, amino, hydroxy, alkoxy, mono(C₁-C₆)alkylamino,di(C₁-C₆)alkylamino, C₂-C₆alkenyl, C₂-C₆alkynyl, C₁-C₆haloalkyl,C₁-C₆haloalkoxy, aromatic heterocyclylalkyl;

R₃ represents hydrogen, C₁-C₃alkyl, or C₃-C₆cycloalkyl.

The compound of formula (I), an isomer, a pharmaceutically acceptablesalt or a solvate thereof according to any item of the first aspect ofthe present invention is provided, wherein:

R₁ represents aromatic heterocyclyl or substituted aromaticheterocyclyl, wherein the substituent is selected from the groupconsisting of halogen, nitro, amino, cyano, C₁-C₆alkoxy, and C₁-C₆alkylthio.

The compound of formula (I), an isomer, a pharmaceutically acceptablesalt or a solvate thereof according to any item of the first aspect ofthe present invention is provided, wherein:

R₁ represents pyrimidinyl or thiazolyl, said pyrimidinyl or thiazolylbeing unsubstituted or substituted with one or two substituentsindependently selected from the group consisting of methyl, halogen,nitro, amino, cyano, methoxy, ethoxy, methylthio and ethylthio.

The compound of formula (I), an isomer, a pharmaceutically acceptablesalt or a solvate thereof according to any item of the first aspect ofthe present invention is provided, wherein:

R₂ represents hydrogen, C₁-C₆alkyl, C₁-C₆alkoxycarbonyl, C₁-C₆alkanoyl,substituted C₁-C₆alkanoyl, 5- or 6-membered aliphatic heterocyclyl,substituted 5- or 6-membered aliphatic heterocyclyl, aryl, substitutedaryl, arylC₁-C₆alkyl, substituted arylC₁-C₆alkyl, arylsulfonyl,substituted arylsulfonyl, aromatic heterocyclyl, substituted aromaticheterocyclyl, aromatic heterocyclylsulfonyl, aromatic heterocyclylacyl,wherein the substituent is selected from the group consisting ofC₁-C₆alkyl, halogen, nitro, cyano, amino, C₁-C₆alkoxy, aromaticheterocyclylC₁-C₃alkyl.

The compound of formula (I), an isomer, a pharmaceutically acceptablesalt or a solvate thereof according to any item of the first aspect ofthe present invention is provided, wherein:

R₃ represents hydrogen, or methyl.

The compound of formula (I), an isomer, a pharmaceutically acceptablesalt or a solvate thereof according to any item of the first aspect ofthe present invention is provided, wherein:

A represents CH₂, O, S,

wherein “

” terminal is attached to R₂;

R₁ represents pyrimidinyl, wherein said pyrimidinyl is unsubstituted orsubstituted with one or two substituents independently selected from thegroup consisting of methyl, halogen, nitro, amino, cyano, methoxy,ethoxy, methylthio and ethylthio;

R₂ represents hydrogen, t-butyl, t-butoxycarbonyl, thiazol-2-sulfonyl,imidazol-1-formyl, phenyl, 1-naphthyl, 2-naphthyl, 4-fluorophenyl,imidazol-1-yl, triazol-1-yl, 2-chloro-benzimidazol-1-yl,2,3-dichlorobenzenesulfonyl, 2,4-chlorobenzenesulfonyl, 3-chlorobenzyl,4-chlorobenzyl, 4-fluorobenzyl, acetyl, trifluoroacetyl,1,2,4-triazol-1-methylpyrrolidin-1-yl;

R₃ represents hydrogen, or methyl.

In an embodiment of the present invention, the compound of formula (I),an isomer, a pharmaceutically acceptable salt or a solvate thereof isselected from the following compounds:

-   (1) t-butyl    N-methyl-N-[1-(4-chloropyrimidin-2-yl)piperidin-4-yl]carbamate;-   (2) t-butyl    N-methyl-N-[1-(2-chloropyrimidin-4-yl)piperidin-4-yl]carbamate;-   (3) t-butyl    N-methylN-[1-(6-chloropyrimidin-4-yl)piperidin-4-yl]carbamate;-   (4) t-butyl    N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate;-   (5) t-butyl    N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate;-   (6) t-butyl    N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate;-   (7) t-butyl    N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate;-   (8)    (1-t-butoxycarbonylpiperidin-4-yl)N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)    piperidin-4-yl]carbamate;-   (9)    (S)-(1-t-butoxycarbonylpyrrolidin-3-yl)N-methyl-N-[1-(4-methylthiopyrimidin-2-yl)    piperidin-4-yl]carbamate;-   (10)    (S)-(pyrrolidin-3-yl)N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate    hydrochloride;-   (11)    [1-(2,4-dichlorobenzenesulfonyl)piperidin-4-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate;-   (12)    (S)-[1-(imidazole-1-carbonyl)pyrrolidin-3-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate;-   (13)    (S)-[1-(2-thienylsulfonyl)pyrrolidin-3-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)    piperidin-4-yl]carbamate;-   (14) p-fluorophenylthio    N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate;-   (15) p-fluorophenylthio    N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate;-   (16)    (1-acetylpiperidin-4-yl)N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate;-   (17)    (S)-[1-(2-thienylsulfonyl)pyrrolidin-3-yl]N-methyl-N-[1-(4-methoxypyrimidin-2-yl)    piperidin-4-yl]carbamate;-   (18)    [1-(3-chlorobenzyl)piperidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)    piperidin-4-yl]carbamate;-   (19)    [1-(3-chlorobenzyl)piperidin-4-yl]N-methyl-N-[1-(4-methoxypyrimidin-2-yl)    piperidin-4-yl]carbamate;-   (20)    [1-(2,3-dichlorobenzenesulfonyl)piperidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate;-   (21)    [1-(2,4-dichlorobenzenesulfonyl)piperidin-4-yl]N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate;-   (22)    [1-(4-fluorobenzyl)piperidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)    piperidin-4-yl]carbamate;-   (23)    (S)-[1-(2,3-dichlorobenzenesulfonyl)pyrrolidin-3-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate;-   (24)    (S)-[1-(2,4-dichlorobenzenesulfonyl)pyrrolidin-3-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate;-   (25)    (1-acetylpiperidin-3-yl)N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate;-   (26)    [1-(2,2,2-trifluoroacetyl)piperidin-3-yl]N-methyl-N-[1-(6-methoxypyrimidin-4-yl)    piperidin-4-yl]carbamate;-   (27)    [1-(imidazole-1-carbonyl)piperidin-3-yl]N-methyl-N-[1-(6-methoxypyrimidin-4-yl)    piperidin-4-yl]carbamate;-   (28)    [1-(2-thienylsulfonyl)piperidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)    piperidin-4-yl]carbamate;-   (29)    [1-(1-thienylsulfonyl)piperidin-4-yl]N-methyl-N-[1-(4-methoxypyrimidin-2-yl)    piperidin-4-yl]carbamate;-   (30)    N-methyl-N-[1-(4-chloropyrimidin-2-yl)piperidin-4-yl]-[2-(imidazol-1-yl)]acetamide;-   (31)    N-methyl-N-[1-(4-chloropyrimidin-2-yppiperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)    acetamide;-   (32)    N-methyl-N-[1-(2-chloropyrimidin-4-yl)piperidin-4-yl]-2-(2-chloro-benzoimidazol-1-yl)acetamide;-   (33)    N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-[2-(imidazol-1-yl)]acetamide;-   (34)    N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)    acetamide;-   (35)    N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-2-(2-chloro-benzoimidazol-1-yl)acetamide;-   (36)    N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]-2-{[3-(1H-1,2,4-triazol-1-methyl)]pyrrolidin-1-yl}acetamide;-   (37)    N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]-2-{[2-(1H-1,2,4-triazol-1-methyl)]pyrrolidin-1-yl}acetamide;-   (38)    N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-2-{[2-(1H-1,2,4-triazol-1-methyl)]pyrrolidin-1-yl}acetamide;-   (39)    N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]-2-{[2-(1H-1,2,4-triazol-1-methyl)]pyrrolidin-1-yl}acetamide;-   (40)    N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)    acetamide;-   (41)    N-methyl-N-[1-(4-methylpyrimidin-2-yl)piperidin-4-yl]-2-(2-chloro-benzoimidazol-1-yl)    acetamide;-   (42)    N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)    acetamide;-   (43)    N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)    acetamide;-   (44)    N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)    acetamide.

In the second aspect, the present invention provides a method forpreparing the compound of formula (I), an isomer, a pharmaceuticallyacceptable salt or a solvate thereof according to any item of the firstaspect of the present invention, which comprises the following steps:

(1) Compound 2 and compound 1 undergo nucleophilic substitution reactionto obtain compound 3; compound 3 is attacked by nucleophilic agent Nu⁻to obtain compound 4; compound 4 undergoes amino deprotection andamidation reaction respectively to obtain an active intermediate 6;wherein, in compound 1, A, B, C represent C or N atom, and at least oneof them is C atom; L is a conventional leaving group such as halogen,—OCOR, —OTs, etc., two Ls may be the same or different, preferably inmeta or para arrangement; Pg is an amino protective group such as Boc;R₁ and R₃ are as defined in claim 1; compound 6 is an amidation productof compound 5, for example, reaction product of compound 5 withchloroacetyl chloride, carbonyl diimidazole or triphosgene, etc.;according to different amidation reagent, compound 6 may be respectivelythe following three forms 6-1, 6-2 or 6-3;

(2) According to different definition of A, the compound of formula (I)as claimed in claim 1 can be prepared by the following stepsrespectively:

When A is CH₂, R₂H is nucleophilic agent, such as amine; R₂H andcompound 6-1 direclty undergo nucleophilic substitution reaction toobtain compound I-1; wherein R₁, R₂, R₃ are as defined in claim 1;

When A is S, R₂SH is nucleophilic agent, such as thiophenol, R₂SH andcompound 6-2 direclty undergo nucleophilic substitution reaction toobtain compound 1-2; wherein R₁, R₂, R₃ are as defined in claim 1; L isa conventional leaving group, such as halogen;

When A is

m=1, 2, n=2, 3; and when m=1, n=2 or 3; and when m=2, n=2; compound 6-3and compound 9 undergo nucleophilic addition reaction to obtain compound7; compound 7 undergoes amino deprotection to obtain compound 8;finally, compound 8 and R₂L undergo nucleophilic substitution reactionto obtain compound 1-3; wherein R₁, R₂, R₃ are as defined in claim 1; Pgis a conventional amino protective group, such as Boc;

The above compounds 3, 4, 5, 7, 8, 9, I-1, I-2 and I-3 are all withinthe scope of the formula (I), and all fall into the protection scope ofthe present invention.

Nucleophilic reagent refers to a reagent having nucleophilicity, such ascompounds having hydroxy, thiol, amino and the like.

Pg is a conventional amino protective group. As to concrete protectionand deprotection methods, please refer to, Green and Woods, <<ProtectiveGroups in Organic Synthesis>>, Chapter 7 “Protection of amino group”.

The preparation method according to the second aspect of the presentinvention, when R₁ is a pyrimidine ring, comprises the followingreaction route, i.e., first, 2,4-dichloro-pyrimidine or4,6-dichloro-pyrimidine as starting material reacts with t-butylN-methyl-N-(piperidin-4-yl)carbamate in a solvent such as THF or DMF, atnormal temperature, in the presence of potassium carbonate, sodiumcarbonate or sodium bicarbonate, to obtain compound 3A; compound 3A thenreacts with sodium methoxide, sodium ethoxide or sodium thiomethoxide inthe corresponding alcohol or THF solvent under refluxing conditions toobtain compound 4A; compound 4A undergoes BOC removal under the actionof trifluoroacetic acid in DCM solvent at room temperature to obtainintermediate 5A; intermediate 5A reacts with CDI in DMF under heatingconditions to obtain compound 6A; compound 6A and the correspondingalcohol dissolved in DMF or DMSO react with compounds 7A-7D respectivelyat room temperature under the catalysis of NaH to obtain compounds8A-8D; compounds 8B, 8C, 8D respectively undergo deprotection under theaction of trifluoroacetic acid in DCM at room temperature to obtaincompounds 9B, 9C, 9D; compounds 9B, 9C, 9D respectively undergocondensation reaction with the corresponding sulfonyl chloride 11 (1) oracid chloride (2) in a solvent such as THF or DMF at room temperature,while neutralizing acid generated during the reaction using potassiumcarbonate, sodium carbonate, sodium bicarbonate, to obtain compounds10(1)B-10(1)D and 10(2)B-10(2)D of the present invention; or

Intermediate 5A dissolved in DCM undergoes condensation reaction with2-chloroacetyl chloride in an ice bath, while neutralizing acidgenerated during the reaction using TEA and the like, to obtain compound12A; compound 12A then reacts with the corresponding amine in a solventsuch as DMF or THF at room temperature, in the presence of potassiumcarbonate, sodium carbonate or sodium bicarbonate, to obtain compound13A of the present invention;

The product can be separated and purified using standard techniques inthe art, such as extraction, chromatography, crystallization anddistillation.

wherein, compounds 11 (1) and 11 (2) mentioned in the above formula arerespectively:

7A-7D, 8B-8D, 9B-9D, 10 (1)B-10 (1)D and 10 (2)B-10 (2)D arerespectively:

A B C  7 R₇—SH

 8

 9

10 (1)

10 (2)

D  7

 8

 9

10 (1)

10 (2)

In the third aspect, the present invention provides a pharmaceuticalcomposition comprising the compound of formula (I), an isomer, apharmaceutically acceptable salt or a solvate thereof according to anyitem of the first aspect of the present invention, and apharmaceutically acceptable carrier or excipient.

The pharmaceutical composition according to any item of the third aspectof the present invention further comprises one or more of otherantitumor drugs, for example, tinib-based antitumor drugs, such asgefitinib, imatinib, imatinib mesylate, nilotinib, sunitinib, lapatinib.

In the fourth aspect, the present invention provides use of the compoundof formula (I), an isomer, a pharmaceutically acceptable salt or asolvate thereof according to any item of the first aspect of the presentinvention in the manufacture of a drug for preventing and/or treatingdrug-resistant tumors or diseases or conditions caused by drug-resistantbacteria.

In the fifth aspect, the present invention provides use of the compoundof formula (I), an isomer, a pharmaceutically acceptable salt or asolvate thereof according to any item of the first aspect of the presentinvention in the manufacture of a drug for preventing and/or treatingtumors, neurodegenerative diseases, allograft rejection, and diseases orconditions related to infection. Preferably, the tumors,neurodegenerative diseases, allograft rejection, and diseases orconditions related to infection are diseases or conditions caused byHeat shock protein 70 (Hsp70).

In the use according to any item of the fourth or fifth aspect of thepresent invention, the tumors are selected from the group consisting ofbreast cancer, prostate cancer, liver cancer, esophageal cancer, gastriccancer, and skin cancer.

In the use according to any item of the fifth aspect of the presentinvention, the neurodegenerative diseases are selected from the groupconsisting of Alzheimer's disease, amyotrophic lateral sclerosis, ataxiatelangiectasia, Creutzfeldt-Jakob disease, Huntington's disease,cerebellar atrophy, multiple sclerosis, Parkinson's disease, primarylateral sclerosis, and spinal muscular atrophy.

In the sixth aspect, the present invention provides a method forpreventing and/or treating drug-resistant tumors or diseases orconditions caused by drug-resistant bacteria, which comprisesadministering to a subject in need of such prevention or treatment aprophylactically and/or therapeutically effective amount of the compoundof formula (I), an isomer, a pharmaceutically acceptable salt or asolvate thereof according to any item of the first aspect of the presentinvention.

In the seventh aspect, the present invention provides a method forpreventing and/or treating tumors, neurodegenerative diseases, allograftrejection, and diseases or conditions related to infection, whichcomprises administering to a subject in need of such prevention ortreatment a prophylactically and/or therapeutically effective amount ofthe compound of formula (I), an isomer, a pharmaceutically acceptablesalt or a solvate thereof according to any item of the first aspect ofthe present invention. Preferably, the tumors, neurodegenerativediseases, allograft rejection, and diseases or conditions related toinfection are diseases or conditions caused by Heat shock protein 70(Hsp70).

In the method according to any item of the sixth or seventh aspect ofthe present invention, the tumors are selected from the group consistingof breast cancer, prostate cancer, liver cancer, esophageal cancer,gastric cancer, and skin cancer.

In the method according to any item of the seventh aspect of the presentinvention, the neurodegenerative diseases are selected from the groupconsisting of Alzheimer's disease, amyotrophic lateral sclerosis, ataxiatelangiectasia, Creutzfeldt-Jakob disease, Huntington's disease,cerebellar atrophy, multiple sclerosis, Parkinson's disease, primarylateral sclerosis, and spinal muscular atrophy.

In the eighth aspect, the present invention provides the compound offormula (I), an isomer, a pharmaceutically acceptable salt or a solvatethereof according to any item of the first aspect of the presentinvention, for preventing and/or treating drug-resistant tumors ordiseases caused by drug-resistant bacteria.

In the ninth aspect, the present invention provides the compound offormula (I), an isomer, a pharmaceutically acceptable salt or a solvatethereof according to any item of the first aspect of the presentinvention, for preventing and/or treating tumors, neurodegenerativediseases, allograft rejection, and diseases or conditions related toinfection. Preferably, the tumors, neurodegenerative diseases, allograftrejection, and diseases or conditions related to infection are diseasesor conditions caused by Heat shock protein 70 (Hsp70).

The compound of formula (I), an isomer, a pharmaceutically acceptablesalt or a solvate thereof according to any item of the eighth or ninthaspect of the present invention, wherein the tumors are selected fromthe group consisting of breast cancer, prostate cancer, liver cancer,esophageal cancer, gastric cancer, and skin cancer.

The compound of formula (I), an isomer, a pharmaceutically acceptablesalt or a solvate thereof according to any item of the ninth aspect ofthe present invention, wherein the neurodegenerative diseases areselected from the group consisting of Alzheimer's disease, amyotrophiclateral sclerosis, ataxia telangiectasia, Creutzfeldt-Jakob disease,Huntington's disease, cerebellar atrophy, multiple sclerosis,Parkinson's disease, primary lateral sclerosis, and spinal muscularatrophy.

In the tenth aspect, the present invention provides a method forresisting/reversing drug resistance of bacteria or drug resistance oftumor cells in cells, which comprises administering to the cells aneffective amount of the compound of formula (I), an isomer, apharmaceutically acceptable salt or a solvate thereof according to anyitem of the first aspect of the present invention.

In the eleventh aspect, the present invention provides a method forinhibiting expression of Heat shock protein 70 (Hsp70) in cells, whichcomprises administering to the cells an effective amount of the compoundof formula (I), an isomer, a pharmaceutically acceptable salt or asolvate thereof according to any item of the first aspect of the presentinvention.

In the method according to any item of the tenth or eleventh aspect ofthe present invention, the cells are cell lines, or cells from asubject.

In the method according to any item of the tenth or eleventh aspect ofthe present invention, the tumor cells are selected from the groupconsisting of breast cancer cells, prostate cancer cells, liver cancercells, esophageal cancer cells, gastric cancer cells, and skin cancercells.

In the method according to any item of the tenth or eleventh aspect ofthe present invention, wherein the method is carried out in vitro.

In the method according to any item of the tenth or eleventh aspect ofthe present invention, wherein the method is carried out in vivo.

In the twelfth aspect, the present invention provides use of thecompound of formula (I), an isomer, a pharmaceutically acceptable saltor a solvate thereof according to any item of the first aspect of thepresent invention in the manufacture of a reagent forresisting/reversing drug resistance of bacteria or drug resistance oftumor cells in cells.

In the thirteenth aspect, the present invention provides use of thecompound of formula (I), an isomer, a pharmaceutically acceptable saltor a solvate thereof according to any item of the first aspect of thepresent invention in the manufacture of a reagent for inhibitingactivity of Heat shock protein 70 (Hsp70) in cells.

In the use according to any item of the twelfth or thirteenth aspect ofthe present invention, the cells are cell lines, or cells from asubject.

In the use according to any item of the twelfth or thirteenth aspect ofthe present invention, the tumor cells are selected from the groupconsisting of breast cancer cells, prostate cancer cells, liver cancercells, esophageal cancer cells, gastric cancer cells, and skin cancercells.

In the use according to any item of the twelfth or thirteenth aspect ofthe present invention, the reagent is used in an in vitro method.

In the use according to any item of the twelfth or thirteenth aspect ofthe present invention, the reagent is used in an in vivo method.

In the fourteenth aspect, the present invention provides the compound offormula (I), an isomer, a pharmaceutically acceptable salt or a solvatethereof according to any item of the first aspect of the presentinvention, for resisting/reversing drug resistance of bacteria or drugresistance of tumor cells in cells.

In the fifteenth aspect, the present invention provides the compound offormula (I), an isomer, a pharmaceutically acceptable salt or a solvatethereof according to any item of the first aspect of the presentinvention, for inhibiting activity of Heat shock protein 70 (Hsp70) incells.

The compound of formula (I), an isomer, a pharmaceutically acceptablesalt or a solvate thereof according to any item of the fourteenth orfifteenth aspect of the present invention, wherein the cells are celllines, or cells from a subject.

The compound of formula (I), an isomer, a pharmaceutically acceptablesalt or a solvate thereof according to any item of the fourteenth orfifteenth aspect of the present invention, wherein the tumor cells areselected from the group consisting of breast cancer cells, prostatecancer cells, liver cancer cells, esophageal cancer cells, gastriccancer cells, and skin cancer cells.

The compound of formula (I), an isomer, a pharmaceutically acceptablesalt or a solvate thereof according to any item of the fourteenth orfifteenth aspect of the present invention, which is used in an in vitromethod.

The compound of formula (I), an isomer, a pharmaceutically acceptablesalt or a solvate thereof according to any item of the fourteenth orfifteenth aspect of the present invention, which is used in an in vivomethod.

In the sixteenth aspect, the present invention provides a kit forresisting/reversing drug resistance of bacteria or drug resistance oftumor cells in cells, which comprises the compound of formula (I), anisomer, a pharmaceutically acceptable salt or a solvate thereofaccording to any item of the first aspect of the present invention, andoptionally instructions.

In the seventeenth aspect, the present invention provides a kit forinhibiting activity of Heat shock protein 70 (Hsp70) in cells, whichcomprises the compound of formula (I), an isomer, a pharmaceuticallyacceptable salt or a solvate thereof according to any item of the firstaspect of the present invention, and optionally instructions.

The terms for describing the present invention occurred in the presentspecification and claims are defined as follows. As to specific terms,if the meaningsthereof defined in the present application areinconsistent with the meanings thereof commonly understood by a personskilled in the art, they have themeaningsdefined in the presentapplication; if not defined in the present application, the terms havethe meanings commonly understood by a person skilled in the art.

The term “alkyl” used herein refers to straight or branched chainmonovalent saturated hydrocarbon group, for example, C₁-C₁₀alkyl,C₁-C₆alkyl, C₁-C₃alkyl. The term “C₁-C₁₀alkyl” refers to straight orbranched chain alkyl having 1 to 10 carbon atoms, e.g., methyl, ethyl,propyl, isopropyl, n-butyl, s-butyl, t-butyl, isobutyl, pentyl,2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl,heptyl and octyl and the like. The term “C₁-C₆alkyl” refers to straightor branched chain alkyl having 1 to 6, i.e., 1, 2, 3, 4, 5 or 6, carbonatoms, typically methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,t-butyl, pentyl and hexyl and the like. The term “C₁-C₃alkyl” refers tostraight or branched chain alkyl having 1, 2 or 3 carbon atoms, i.e.methyl, ethyl, n-propyl and isopropyl. In the present invention, alkylis preferably C₁₋₆ alkyl.

The term “C₂-C₆alkenyl” used herein refers to alkenyl having 2 to 6carbon atoms. The alkenyl has 1, 2 or 3 carbon-carbon double bonds. Whenthere is more than one carbon-carbon double bond, the carbon-carbondouble bonds are conjugated or non-conjugated. Examples of C₂-C₆alkenylin the present invention include vinyl, propenyl.

The term “C₂-C₆alkynyl” used herein refers to alkynyl having 2 to 6carbon atoms. The alkynyl has 1, 2 or 3 carbon-carbon triple bonds. Whenthere is more than one carbon-carbon triple bond, the carbon-carbontriple bonds are conjugated or non-conjugated. Examples of C₂-C₆alkynylin the present invention include ethynyl, propynyl.

The term “halogen” used herein refers to fluorine, chlorine, bromine andiodine atoms.

The term “aryl” used herein refers to an optionally substitutedmonocyclic or bicyclic unsaturated aromatic system having at least oneunsaturated aromatic ring, preferably aryl having 6 to 10, i.e., 6, 7,8, 9 or 10 carbon atoms. Examples of aryl in the present inventioninclude phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl and indenyl and thelike. In the present invention, aryl may be substituted with thefollowing groups: C₁-C₆alkyl, C₁-C₆alkoxy, cyano, halo, hydroxy, amino,nitro, mono(C₁-C₆)alkylamino, di(C₁-C₆)alkylamino, C₂-C₆alkenyl,C₂-C₆alkynyl, C₁-C₆haloalkyl or C₁-C₆haloalkoxy. Specific examplesinclude, but are not limited to, 4-fluorophenyl.

Wherein, “alkyl” as defined above is substituted with one or more of thearyl as defined above, or sulfonyl, acyl are respectively substitutedwith the aryl as defined above, to obtain the aforesaid “arylalkyl”,“arylsulfonyl”, “arolyl” respectively.

Wherein, “alkyl” as defined above is substituted with one or more of thesubstituted aryl as defined above, or sulfonyl, acyl are respectivelysubstituted with the substituted aryl as defined above, to obtain theaforesaid “substituted arylalkyl”, “substituted arylsulfonyl”,“substituted arolyl” respectively. Wherein, the arylalkyl is, forexample, arylC₁-C₃alkyl.

In the present invention, specific examples of the “arylalkyl”,“arylsulfonyl”, “arolyl”, “substituted arylalkyl”, “substitutedarylsulfonyl” or “substituted arolyl” include, but are not limited to,benzyl, phenethyl, 3-chlorobenzyl, 4-chlorobenzyl, 4-fluorobenzyl,2,3-dichlorobenzenesulfonyl, 2,4-dichlorobenzenesulfonyl.

The term “aromatic heterocyclyl” used herein refers to an optionallysubstituted monocyclic or bicyclic unsaturated aromatic ring systemcontaining at least one, for example, 1, 2, 3 or 4 heteroatomsindependently selected from N, O or S, preferably aromatic heterocyclylhaving 5 to 10, i.e., 5, 6, 7, 8, 9 or 10 ring atoms. Examples of the“aromatic heterocyclyl” include, but are not limited to, thienyl,pyridyl, thiazolyl, isothiazolyl, furanyl, pyrrolyl, triazolyl,imidazolyl, triazinyl, diazolyl, oxazolyl, isoxazolyl, pyrazolyl,imidazolonyl, oxazolyl, thiazolonyl, tetrazolyl, thiadiazolyl,benzimidazolyl, benzoxazolyl, benzothiazolyl, tetrahydrotriazolopyridyl,tetrahydrotriazolopyrimidinyl, benzofuranyl, benzothienyl, thioindenyl,indolyl, isoindolyl, pyridonyl, pyridazinyl, pyrazinyl, pyrimidinyl,quinolinyl, phthalazinyl, quinoxalinyl, quinazolinyl, imidazopyridyl,oxazolopyridyl, thiazolopyridyl, imidazopyridazinyl, oxazolopyridazinyl,thiazolopyridazinyl, pteridinyl, furazanyl, benzotriazolyl,pyrazolopyridyl and purinyl and the like.

The term “aliphatic heterocyclyl” used herein refers to an optionallysubstituted monocyclic or bicyclic saturated or partially saturated ringsystem containing at least one and up to four, for example, 1, 2, 3 or 4heteroatom independently selected from N, O or S, preferably aliphaticheterocyclyl having 4 to 10, i.e., 4, 5, 6, 7, 8, 9 or 10 ring atoms,with the proviso that the ring of the heterocyclyl does not contain twoadjacent O or S atoms. Preferred aliphatic heterocyclyl includes, but isnot limited to, saturated cycloalkyl containing at least one, forexample, 1, 2 or 3 heteroatom independently selected from N, O or S,preferably aliphatic heterocyclyl having 3 to 8, i.e., 3, 4, 5, 6, 7 or8 ring atoms. Examples of the “aliphatic heterocyclyl” include, but arenot limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, piperidinyl, morpholinyl or piperazinyl and the like.

In the present invention, aromatic heterocyclyl or aliphaticheterocyclyl may be substituted with the following groups: C₁-C₆alkyl,C₁-C₆alkoxy, cyano, halogen, hydroxy, amino, nitro,mono(C₁-C₆)alkylamino, di(C₁-C₆)alkylamino, C₂-C₆alkenyl, C₂-C₆alkynyl,C₁-C₆haloalkyl, C₁-C₆haloalkoxy or aromatic heterocyclylalkyl orsubstituted aromatic heterocyclylalkyl, to obtain the aforesaid“substituted aromatic heterocyclyl” or “substituted aliphaticheterocyclyl”. Specific examples include, but are not limited to,2-chloropyrimidin-4-yl, 4-chloropyrimidin-2-yl, 6-chloropyrimidin-4-yl,2-methoxypyrimidin-4-yl, 4-methoxypyrimidin-2-yl,6-methoxypyrimidin-4-yl, 2-methylthiopyrimidin-4-yl,4-methylthiopyrimidin-2-yl, 6-methylthiopyrimidin-4-yl,1,2,4-triazol-1-methyl, 2-methylpyrimidin-4-yl, 4-methylpyrimidin-2-yl,6-methylpyrimidin-4-yl, 2-chlorobenzoimidazol-1-yl,tetrahydropyranylmethyl.

Wherein, “alkyl” as defined above is substituted with one or more of thearomatic heterocyclyl as defined above, or sulfonyl, acyl arerespectively substituted with the aromatic heterocyclyl as definedabove, to obtain the aforesaid “aromatic heterocyclylalkyl”, “aromaticheterocyclylsulfonyl”, “aromatic heterocyclylacyl” respectively.

When “alkyl” as defined above is substituted with one or more of thesubstituted aromatic heterocyclyl as defined above, or sulfonyl, acylare respectively substituted with the substituted aromatic heterocyclylas defined above, to obtain the aforesaid “substituted aromaticheterocyclylalkyl”, “substituted aromatic heterocyclylsulfonyl”,“substituted aromatic heterocyclylacyl” respectively. Wherein, thearomatic heterocyclylalkyl is, for example, 5- or 6-membered aromaticheterocyclylC₁-C₃alkyl.

In the present invention, specific examples of the “aromaticheterocyclylalkyl”, “aromatic heterocyclylsulfonyl”, “aromaticheterocyclylacyl”, “substituted aromatic heterocyclylalkyl”,“substituted aromatic heterocyclylsulfonyl” or “substituted aromaticheterocyclylacyl” include, but are not limited to, pyridylethyl,imidazol-1-formyl, thiazol-2-sulfonyl.

The term “C₃-C₆cycloalkyl” used herein refers to a saturated carbocyclicgroup having 3 to 6, i.e., 3, 4, 5 or 6 carbon atoms. The cycloalkyl maybe monocyclic or polycyclic fused system, and can be fused to anaromatic ring. Examples of such groups include cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl and the like. Cycloalkyl herein may beunsubstituted or substituted with appropriate group at one or moresubstitutable positions. For example, in the present invention, thecycloalkyl can be optionally substituted with the following groups:C₁-C₆alkyl, C₁-C₆alkoxy, cyano, halogen, hydroxy, amino, nitro,mono(C₁-C₆)alkylamino, di(C₁-C₆)alkylamino, C₂-C₆alkenyl, C₂-C₆alkynyl,C₁-C₆haloalkyl or C₁-C₆haloalkoxy.

The term “pharmaceutically acceptable salts” used herein refers to saltsof compounds of the present invention which are pharmaceuticallyacceptable and have the desired pharmacological activity of the parentcompounds. The salts include: acid addition salts formed with inorganicacids or organic acids, wherein the inorganic acids are such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like; and the organic acids are such as aceticacid, propionic acid, hexanoic acid, cyclopentyl propionic acid,glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid,malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid,camphor sulfonic acid, glucoheptonic acid, gluconic acid, glutamic acid,hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid andthe like; or salts formed when acidic protons present on the parentcompoundsare substituted with metal ions, such as alkali metal ions oralkaline earth metal ions; or coordination compounds formed with organicbase, wherein the organic base is such as ethanolamine, diethanolamine,triethanolamine, N-methyl glucosamine and the like.

The term “solvate” used herein refers to a substance formed by combiningthe compound of the present invention with a pharmaceutically acceptablesolvent. The pharmaceutically acceptable solvent includes water,ethanol, acetic acid and the like. The solvate includes stoichiometricamount of solvate and non-stoichiometric amount of solvate, and ispreferably hydrate. The compound of the present invention may becrystallized or recrystallized with water or various organic solvents.In this case, various solvates may be formed.

The term “subject” used herein includes mammals and human, preferablyhuman.

Those skilled in the art will appreciate that the compound of theinvention has stereoisomerism, for example, cis- and trans-isomers orenantiomers. Therefore, when the compound of the present invention ismentioned in the present specification, the compound of the presentinvention includes a compound of formula (I), an isomer, apharmaceutically acceptable salt or a solvate thereof. The compound ofthe present invention also includes active metabolite of the compound ofthe present invention in mammals.

The present specification illustrates in the part “Mode of carrying outthe invention” the preparation method of the compound of the presentinvention and the anti-tumor effect of the same.

The pharmaceutical composition of the present invention comprises aneffective amount of the compound of formula (I), an isomer, apharmaceutically acceptable salt or a solvate thereof, and one or morepharmaceutically acceptable carrier. The pharmaceutically acceptablecarrier herein includes, but is not limited to: ion exchangers, alumina,aluminum stearate, lecithin, serum proteins such as human serum albumin,buffers such as phosphates, glycerine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinylpyrrolidone,cellulose-based materials, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylate, beeswax, lanolin.

The pharmaceutical composition comprising the compound of the presentinvention can be administered according to any of the following routes:oral, spray inhalation, rectal, nasal, buccal, topical, parenteral suchas subcutaneous, intravenous, intramuscular, intraperitoneal,intrathecal, intraventricular, intrasternal or intracranial injection orinfusion, or by means of an explanted reservoir, wherein oral,intraperitoneal or intravenous administration route is preferred.

In the case of oral administration, the compound of the presentinvention may be made into any orally acceptable formulation form,including but not limited to tablets, capsules, aqueous solutions oraqueous suspensions. Wherein, the carrier used in tablets generallyincludes lactose and corn starch, additionally, a lubricant such asmagnesium stearate may also be added. The diluent used in capsulesgenerally includes lactose and dried corn starch. Aqueous suspensionsaregenerally used by mixing active ingredient with suitable emulsifier andsuspending agent. If desired, some sweetening agents, flavoring agentsor coloring agents may also be added in the aboveoral formulation forms.

In the case of topical administration, especially for the treatment ofaffected surfaces or organs where topical application is easy to reach,such as eyes, skin, or lower intestinal neurological diseases, thecompound of the present invention may be made into different topicalformulation forms according to different affected surfaces or organs,which are specifically described as follows:

In the case of topical ocular administration, the compound of thepresent invention may be formulated into the formulation form ofmicronised suspension or solution, and the carrier used is isotonicsterile saline at a certain pH, to which a preservative such as benzylchloride alkoxide may be added or not. For ocular administration, thecompound may also be made into an ointment form such as vaselineointment.

In the case of topical dermal administration, the compound of thepresent invention may be made into suitable formulation forms ofointments, lotions or creams, wherein active ingredient is suspended ordissolved in one or more carriers. The carrier that may be used inointments includes, but is not limited to: mineral oil, liquidpetrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene, emulsifying wax and water; the carrier that may beused in lotions or creams includes, but is not limited to: mineral oil,sorbitanmonostearate, Tween 60, cetyl wax, hexadecene aromatic alcohols,2-octyldodecanol, benzyl alcohol and water.

The compound of the present invention may also be administered insterile injectable formulation forms, including sterile injectableaqueous or oil suspensions or sterile injectable solutions. The carrierand solvent that may be used therein include water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile non-volatileoils, such as mono- or diglycerides, may also be used as solvent orsuspending medium.

The dose of the compound of the present invention administred to thesubject depends on the type and severity of the disease or condition andthe characteristics of the subject, for example, general healthcondition, age, gender, body weight and tolerance to drugs, and alsodepends on the type of formulation and the administration route of drug,and the period or interval of administration, etc. Those skilled in theart can determine an appropriate dose according to these factors andother factors. Generally, the daily dose of the compound of the presentinvention useful for treating tumor may be about 1-800 mg, and thisdaily dose may be administered one or more times according to thespecific condition. The compound of the present invention may beprovided in dosage unit, and the content of the compound in the dosageunit may be 0.1-200 mg, e.g., 1-100 mg.

Beneficial Effects of the Present Invention

The present invention provides a type of piperidine carboxamide compoundhaving novel structure, which can effectively inhibit/reverse drugresistance of bacteria or tumors, and can act as Hsp70 inhibitior toeffectively prevent an/or treat diseases or conditions caused by Hsp70.This provides a new medical strategy for the treatment of relateddiseases.

Mode of Carrying Out the Invention

The embodiments of the present invention will be described in detail incombination with the following examples. However, as will be appreciatedby a person skilled in the art, the following examples are only used toillustrate the present invention, and should not be construed aslimiting the scope of the present invention. The examples, in which thespecific conditions are not indicated, are carried out according toconventional conditions or the conditions recommended by themanufacturer. The reagents or instruments, for which the manufacturersare not indicated, are all commercially available conventional products.

Melting point of the compound is measured using RY-1 melting pointapparatus, with thermometer not corrected. Mass spectrum is measuredusing Micromass ZabSpec high resolution mass spectrometer (resolution1000). ¹H NMR is measured using JNM-ECA-400 superconducting NMRinstrument, working frequency ¹H NMR 300 MHz, ¹³C NMR 100 MHz.

EXAMPLE 1 t-butylN-methyl-N-[1-(4-chloropyrimidin-2-yl)piperidin-4-yl]carbamate

75 g of 2,4-dichloropyrimidine, 129 g of4-N-t-butoxycarbonyl-4-N-methylaminopiperidine, 75 g of TEA and THF (700ml, 10×) were added to a three-necked round-bottom flask, and reacted atroom temperature. the reaction was completed after 2h, and the reactionsolution was evaporated under reduced pressure, followed by extractingwith DCM and water twice, and washing with saturated saline solutiontwice. The organic phases were combined, dried with anhydrous sodiumsulfate, and concentrated to obtain 190 g of a crude product. 500 mg ofthe crude product was separated through a chromatographic column, togive a product having less polarity, which was the target compoundt-butyl N-methyl-N-[1-(4-chloropyrimidin-2-yl)piperidin-4-yl]carbamateof Example 1, and a product having more polarity, which was the targetcompound t-butylN-methyl-N-[1-(2-chloropyrimidin-4-yl)piperidin-4-yl]carbamate ofExample 2. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.49 (s, 9H); δ 1.62-1.69 (m,2H); δ 1.74-1.73 (t, 2H); δ 2.73 (s, 3H); δ 2.89-2.95 (m, 2H); δ4.28-4.32 (m, 2H); δ 4.86-4.90 (m, 2H); δ 6.49-6.51 (d, 1H); δ 8.15-8.16(d, 1H). MS (TOF) 326.85 (M+).

EXAMPLE 2 t-butylN-methyl-N-[1-(2-chloropyrimidin-4-yl)piperidin-4-yl]carbamate

According to the method of Example 1, a product having more polarity wasseparated, which was the compound of Example 2. ¹H-NMR (300 MHz,CDCl₃-d) δ 1.495 (s, 9H); δ 1.60-1.70 (m, 2H); δ 1.79-1.82 (t, 2H); δ2.73 (s, 3H); δ 2.94-3.00 (t, 2H); δ 4.20-4.30 (m, 2H); δ 4.50-4.54 (m,2H); δ 6.43-6.44 (d, 1H); δ 8.04-8.06 (d, 1H). MS (TOF) 326.85 (M+).

EXAMPLE 3 t-butylN-methyl-N-[1-(6-chloropyrimidin-4-yl)piperidin-4-yl]carbamate

The method of Example 1 was carried out, except for the use of4,6-dichloropyrimidine in place of 2,4-dichloropyrimidine, to obtain 50g of the product. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.47 (s, 9H); δ 1.60-1.61(m, 2H); δ 1.63-1.64 (m, 2H); δ 2.79 (s, 3H); δ 2.91-2.97 (t, 2H); δ4.25 (s, 1H); δ 4.48 (s, 2H); δ 6.52 (s, 1H); δ 8.36 (s, 1H). MS (TOF)326.8 (M+).

EXAMPLE 4 t-butylN-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate

190 g of the crude product obtained in Example 1 was dissolved in 800 mlof anhydrous methanol, to which 35 g (1.2 eq) of sodium methoxide wasadded slowly to carry out an exothermic reaction. T=70° C. and thereaction was completed after 6h. The reaction solution was evaporatedunder reduced pressure, followed by extracting with DCM and water twice.The organic phases were combined, dried, concentrated and separatedthrough a chromatographic column, to obtain 90 g of the target compound.¹H-NMR (300 MHz, CDCl₃-d) δ 1.46 (s, 9H); δ 1.60-1.72 (m, 4H); δ 2.70(s, 3H); δ 2.82-2.90 (t, 2H); δ 3.88 (s, 3H); δ 4.84-4.89 (d, 1H); δ5.95-5.96 (d, 1H); δ 8.02-8.04 (d, 1H). MS (TOF) 322.4 (M+).

EXAMPLE 5 t-butylN-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate

According to the method of Example 4, a product, which had less polaritythan that of the product of Example 4, was separated through thechromatographic column, which was the target compound of Example 5, 40g. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.46 (s, 9H); δ 1.62 (m, 2H); δ 1.72 (m,2H); δ 2.70 (s, 3H); δ 2.90 (t, 2H); δ 3.91 (s, 3H); δ 4.49-4.51 (m,3H); δ 6.18-6.19 (m, 2H); δ 8.00-8.01 (d, 1H). MS (TOF) 322.4 (M+).

EXAMPLE 6 t-butylN-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate

The method of Example 4 was carried out, with the use of the productobtained in Example 2 as starting material, and the use of sodiumthiomethoxide in place of sodium methoxide, to obtain 36 g of the targetcompound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.494 (s, 9H); δ 1.615-1.651 (m,2H); δ 1.752-1.785 (m, 2H); δ 2.521 (s, 3H) δ 2.724 (s, 3H); δ2.892-2.953 (t, 2H); δ 4.200-4.529 (m, 2H); δ 4.532-4.569 (m, 2H); δ6.223-6.239 (d, 1H); δ 8.025-8.040 (d, 1H). MS (TOF) 338.5 (M+).

EXAMPLE 7 t-butylN-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate

The method of Example 4 was carried out, with the use of the compound asdescribed in Example 3 as starting material, to obtain 33 g of thetarget compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.47 (s, 9H); δ 1.55-1.65(m, 2H); δ 1.72 (m, 2H); δ 2.70 (s, 3H); δ 2.89 (t, 2H); δ 3.91 (s, 3H);δ 4.20 (m, 1H); δ 4.42-4.45 (d, 2H); δ 5.84 (s, 1H); δ 8.32 (s, 1H). MS(TOF) 322.4 (M+).

EXAMPLE 8 (1-t-butoxycarbonylpiperidin-4-yl)N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate

The compound of Example 6 was added to concentrated hydrochloric acidfor the removal of Boc, followed by the addition of potassium carbonateto obtain a free amine. 20 g (1 eq) of the resulting free amine wasadded to 200 ml of anhydrous DCM, to which 17 g (1.2 eq) of CDI wasadded with stirring. the reaction was completed after reacting at roomtemperature for 2h, and the reaction solution was evaporated underreduced pressure, which was then dissolved in 200 ml DMF. 17 g oft-butyl 4-hydroxy-1-piperidinyl formate as starting material wasdissolved in 200 ml DMF, to which 20 g NaH was added slowly; and theresulting solution was added dropwise to the above reaction solutionafter stirred for 30 min. the reaction was completed after 1h, and thereaction solution was extracted with DCM and water twice. The organicphase was washed with saturated saline solution twice, dried,concentrated and separated through a chromatographic column, to obtain10 g of a pure product. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.4642 (s, 9H); δ1.62-1.74 (m, 8H); δ 2.50 (s, 3H); δ 2.75 (s, 3H); δ 2.88-2.96 (t, 2H);δ 3.25-3.29 (m, 2H); δ 3.63-3.65 (m, 2H); δ 4.51-4.55 (m, 2H); δ4.88-5.05 (m, 1H); δ 6.21-6.25 (d, 1H); δ 8.01-8.06 (d, 1H). MS (TOF)465.6 (M+).

EXAMPLE 9 (S)-(1-t-butoxycarbonylpyrrolidin-3-yl)N-methyl-N-[1-(2-methylthiopyrimidin-4-yl) piperidin-4-yl]carbamate

The method of Example 4 was carried out, with the use of sodiumthiomethoxide in place of sodium methoxide, to obtain t-butylN-methyl-N-[1-(4-methylthiopyrimidin-2-yl)piperidin-4-yl]carbamate. Themethod of Example 8 was carried out, with the use of the productobtained above as starting material, and the use of t-butyl(S)-3-hydroxypyrrolidin-1-yl formate in place of t-butyl4-hydroxy-1-piperidinyl formate as starting material, to obtain 5 g ofthe product. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.47 (s, 9H); δ 1.62-1.71 (m,5H); δ 2.05 (m, 2H); δ 2.72 (s, 3H); δ 2.79-2.90 (m, 2H); δ 3.41-3.45(m, 4H); δ 3.90 (s, 3H); δ 4.87-4.91 (m, 2H); δ 5.26-5.30 (m, 1H); δ5.26-5.30 (m, 1H); δ 5.98-5.99 (d, 1H); δ 8.04-8.06 (d, 1H). MS (TOF)435.5 (M+).

EXAMPLE 10 (S)-(pyrrolidin-3-yl)N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamatehydrochloride

The product obtained in Example 6 was subject in sequence to the methodof Example 8, and the removal of Boc, to obtain 6 g of the compound ofExample 10, in which t-butyl (S)-3-hydroxypyrrolidin-1-yl formate wasused in place of t-butyl (S)-4-hydroxy-1-piperidinyl formate as startingmaterial. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.099 (t, 1H); δ 1.685-1.787 (m,4H); δ 2.208 (m, 2H); δ 2.686 (s, 3H); δ 2.977-3.042 (m, 1H); δ3.400-3.422 (m, 1H); δ 3.459-3.562 (m, 5H); δ 4.118-4.153 (m, 2H); δ4.712 (s, 3H); δ 5.124-5.156 (m, 1H); δ 5.288-5.294 (m, 1H); δ6.645-6.664 (d, 1H); δ 7.761-7.780 (d, 1H). MS (TOF) 387.9 (M+).

EXAMPLE 11[1-(2,4-dichlorobenzenesulfonyl)piperidin-4-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate

400 mg of the compound obtained in Example 8 was subject to the removalof BOC, followed by the addition of K₂CO₃ to obtain a free amine. Theresulting free amine was dissolved in 3 ml THF, to carry out reaction atroom temperature, with the addition of 0.5 g K₂CO₃, and 227 mg of2,4-dichlorobenzenesulfonyl chloride. The reaction was completed after4h, and a small amount of water and ethyl acetate were added, followedby extracting with ethyl acetate three times, and washing with saturatedsodium chloride solution twice. The organic phases were combined, driedwith anhydrous sodium sulfate, concentrated and separated through achromatographic column, to obtain 130 mg of the target compound. ¹H-NMR(300 MHz, CDCl₃-d) δ 1.64-1.81 (m, 6H); δ 1.96-2.05 (m, 2H); δ 2.50 (s,3H); δ 2.72 (s, 3H); δ 2.91 (m, 2H); δ 3.26 (m, 2H); δ 3.51 (m, 2H); δ4.30 (m, 1H); δ 4.51 (m, 2H); δ 4.86-4.87 (m, 1H); δ 6.20-6.22 (d, 1H);δ 7.26-7.36 (dd, 1H); δ 7.54-7.55 (d, 1H); δ 8.00 (s, 1H); δ 8.02-8.03(t, 1H). MS (TOF) 574.5 (M+).

EXAMPLE 12(S)-[1-(imidazole-1-carbonyl)pyrrolidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate

The method of Example 10 was carried out, with the use of the productobtained in Example 5 as starting material. 20 g (1 eq) of the resultingBOC-removed product was added to 200 ml anhydrous DCM, to which 17 g(1.2 eq) of CDI was added with stirring, followed by reacting at roomtemperature for 2 h, and separating to obtain 20 g of the product.¹H-NMR (300 MHz, CDCl₃-d) δ 1.24-1.28 (m, 4H); δ 1.65-1.74 (d, 1H); δ2.05-2.23 (d, 1H); δ 2.73 (s, 3H); δ 2.94 (m, 2H); δ 3.70-3.78 (m, 4H);δ 3.93 (s, 3H); δ 4.3 (m, 1H); δ 4.53 (m, 2H); δ 5.30-5.35 (m, 1H); δ6.20-6.35 (d, 1H); δ 7.11 (s, 3H); δ 7.36 (s, 3H); δ 8.03-8.05 (m, 2H).MS (TOF) 429.5 (M+).

EXAMPLE 13(5)-[1-(2-thienylsulfonyl)pyrrolidin-3-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate

The method of Example 11 was carried out, with the use of2-thienylsulfonyl chloride in place of 2,4-dichlorobenzenesulfonylchloride, to obtain 150 mg of the target compound. ¹H-NMR (300 MHz,CDCl₃-d) δ 1.54-1.68 (m, 4H); δ 2.04-2.07 (m, 2H); δ 2.42 (s, 2H); δ2.50 (s, 2H); δ 2.75-2.90 (m, 3H); δ 3.36-3.54 (m, 4H); δ 4.24 (m, 1H);δ 4.50 (m, 1H); δ 5.15-5.30 (m, 1H); δ 6.20-6.23 (d, 1H); δ 7.14 (t,1H); δ 7.60-7.61 (m, 2H); δ 8.01-8.03 (d, 1H). MS (TOF) 497.7 (M+).

EXAMPLE 14 p-fluorophenylthioN-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate

The product obtained in Example 5 was subject to the removal of Boc. 1 gof the resulting product was dissolved in 20 ml of anhydrous DCM, towhich a suitable amount of 1 ml TEA was added with stirring. Inice-water bath, 1 g of triphosgene was dissolved in 5 ml of anhydrousDCM, and then the triphosgene solution was added dropwise slowly intothe solution obtained above, with stirring continuously. After reactingfor 1 h, 1 eq of p-aminothiophenol and 1 ml triethanolamine were addedto continue the reaction at room temperature. The reaction was completedafter 2h, and 20 ml of water was added, followed by extracting with DCMthree times, and washing with saturated sodium chloride solution twice.The organic phases were combined, dried with anhydrous sodium sulfate,concentrated and separated through a chromatographic column, to obtain900 mg of the target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.73-1.85 (m,5H); δ 2.77 (s, 5H); δ 3.96 (s, 3H); δ 4.57 (s, 3H); δ 6.23-6.24 (d,1H); δ 7.09-7.13 (m, 2H); δ 7.47-7.51 (m, 2H); δ 8.05-8.07 (d, 1H). MS(TOF) 376.4 (M+).

EXAMPLE 15 p-fluorophenylthioN-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate

The method of Example 14 was carried out, with the use of the productobtained in Example 6 as starting material, to obtain 550 mg of thetarget compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.72-1.84 (m, 5H); δ 2.53(s, 3H); δ 2.94 (s, 5H); δ 2.67 (m, 3H); δ 6.24-6.25 (d, 1H); δ7.09-7.13 (m, 2H); δ 7.47-7.51 (m, 2H); δ 8.05-8.06 (d, 1H). MS (TOF)392.5 (M+).

EXAMPLE 16 (1-acetylpiperidin-4-yl)N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate

The methods of Example 8 and Example 11 were respectively carried out,with the use of the product obtained in Example 4 as starting material,and the use of acetyl chloride in place of 2,4-dichlorobenzenesulfonylchloride in Example 11, to obtain 160 mg of the target compound. ¹H-NMR(300 MHz, CDCl₃-d) δ 1.66-1.71 (m, 6H); δ 1.92 (m, 2H); δ 2.11 (s, 3H);δ 2.77 (s, 3H); δ 2.89 (m, 2H); δ 3.37-3.84 (m, 4H); δ 3.88 (s, 3H); δ4.30 (m, 1H); δ 4.88-4.91 (m, 3H); δ 5.97-5.98 (d, 1H); δ 8.04-8.05 (d,1H). MS (TOF) 392. 5 (M+).

EXAMPLE 17(S)-[1-(2-thienylsulfonyl)pyrrolidin-3-yl]N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate

The method of Example 11 was carried out, with the use of the productobtained in Example 9 as starting material, and the use of2-thienylsulfonyl chloride in place of 2,4-dichlorobenzenesulfonylchloride, to obtain 170 mg of the target compound. ¹H-NMR (300 MHz,CDCl₃-d) δ 1.58-1.66 (m, 4H); δ 2.03-2.08 (m, 2H); δ 2.44 (s, 2H); δ3.40-3.60 (m, 4H); δ 3.90 (s, 3H); δ 4.87-4.90 (d, 2H); δ 5.16 (s, 1H);δ 5.99-6.00 (d, 1H); δ 7.15 (m, 1H); δ 7.61-7.62 (t, 1H); δ 8.05-8.06(d, 1H). MS (TOF) 481.6 (M+).

EXAMPLE 18[1-(3-chlorobenzyl)piperidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate

The methods of Example 8 and Example 11 were respectively carried out,with the use of the product obtained in Example 5 as starting material,the use of t-butyl 3-hydroxy-1-piperidinyl formate in place of t-butyl4-hydroxy-1-piperidinyl formate in Example 8 as starting material, andthe use of 3-chlorobenzyl chloride in place of2,4-dichlorobenzenesulfonyl chloride in Example 11, to obtain 150 mg ofthe target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.74-2.76 (m, 12H); δ2.93 (s, 3H); δ 3.46 (m, 2H); δ 3.92 (s, 3H); δ 4.50-4.52 (m, 2H); δ4.81 (s, 1H); δ 5.30 (s, 1H); δ 6.19-6.20 (d, 1H); δ 7.21-7.22 (m, 2H);δ 7.33 (s, 1H); δ 8.01-8.02 (d, 1H). MS (TOF) 474 (M+).

EXAMPLE 19[1-(3-chlorobenzyl)piperidin-4-yl]N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate

The methods of Example 8 and Example 11 were respectively carried out,with the use of the product obtained in Example 4 as starting material,and the use of 3-chlorobenzyl chloride in place of2,4-dichlorobenzenesulfonyl chloride in Example 11, to obtain 145 mg ofthe target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.65-1.80 (m, 6H); δ1.95-1.98 (m, 2H); δ 2.36 (m, 2H); δ 2.65 (m, 2H); δ 2.66 (s, 2H); δ2.77 (m, 2H); δ 3.51 (s, 2H); δ 3.90 (s, 2H); δ 4.79 (s, 1H); δ4.89-4.92 (d, 2H); δ 5.98-5.99 (d, 1H); δ 7.23-7.28 (m, 3H); δ 7.36 (s,1H); δ 8.05-8.07 (d, 1H). MS (TOF) 474 (M+).

EXAMPLE 20[1-(2,3-dichlorobenzenesulfonyl)piperidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate

The methods of Example 8 and Example 11 were respectively carried out,with the use of the product obtained in Example 5 as starting material,the use of t-butyl 3-hydroxy-1-piperidinyl formate in place of t-butyl4-hydroxy-1-piperidinyl formate in Example 8 as starting material, andthe use of 2,3-dichlorobenzenesulfonyl chloride in place of2,4-dichlorobenzenesulfonyl chloride in Example 11, to obtain 156 mg ofthe target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.61-1.95 (m, 9H); δ2.75-2.78 (m, 3H); δ 2.95 (m, 3H); δ 3.29 (m, 1H); δ 3.54 (d, 1H); δ3.92 (m, 3H); δ 4.83 (m, 1H); δ 6.19-6.21 (d, 1H); δ 7.28-7.35 (m, 1H);δ 7.64-7.66 (d, 1H); δ 8.00-8.03 (d, 1H). MS (TOF) 558. 5 (M+).

EXAMPLE 21[1-(2,4-dichlorobenzenesulfonyl)piperidin-4-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate

The methods of Example 8 and Example 11 were respectively carried out,with the use of the product obtained in Example 4 as starting material,to obtain 151 mg of the target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ1.71-1.98 (m, 8H); δ 2.76 (s, 3H); δ 2.90 (s, 2H); δ 3.52 (m, 2H); δ3.91 (s, 3H); δ 4.89-4.92 (d, 3H); δ 5.99-6.00 (d, 1H); δ 7.28-7.29 (d,1H); δ 7.56 (s, 1H); δ 8.02-8.07 (m, 2H). MS (TOF) 558.5 (M+).

EXAMPLE 22[1-(4-fluorobenzyl)piperidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate

The methods of Example 8 and Example 11 were respectively carried out,with the use of the product obtained in Example 5 as starting material,and the use of 4-fluorobenzyl chloride in place of2,4-dichlorobenzenesulfonyl chloride in Example 11, to obtain 147 mg ofthe target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.63-2.23 (m, 10H); δ2.55 (m, 1H); δ 2.76 (s, 3H); δ 2.77 (m, 1H); δ 2.93 (t, 2H); δ 3.50 (m,2H); δ 4.20-4.81 (m, 4H); δ 6.20-6.21 (d, 1H); δ 6.93-7.02 (t, 2H); δ7.26-7.30 (m, 2H); δ 8.04-8.05 (d, 1H). MS (TOF) 457.5 (M+).

EXAMPLE 23(S)-[1-(2,3-dichlorobenzenesulfonyl)pyrrolidin-3-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate

The method of Example 11 was carried out, with the use of the compoundwhich has not been subject to the removal of Boc obtained in Example 10as starting material, and the use of 2,3-dichlorobenzenesulfonylchloride in place of 2,4-dichlorobenzenesulfonyl chloride, to obtain 165mg of the target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.50-1.80 (m,4H); δ 2.18 (m, 2H); δ 2.52 (s, 3H); δ 2.65 (s, 3H); δ 2.93 (t, 2H); δ3.60 (m, 4H); δ 4.20-4.30 (m, 1H); δ 4.54 (s, 2H); δ 5.31 (s, 1H); δ6.22-6.23 (d, 1H); δ 7.28-7.36 (t, 1H); δ 8.03-8.07 (d, 1H). MS (TOF)560.5 (M+).

EXAMPLE 24(S)-[1-(2,4-dichlorobenzenesulfonyl)pyrrolidin-3-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate

The method of Example 11 was carried out, with the use of the compoundwhich has not been subject to the removal of Boc obtained in Example 10as starting material, to obtain 149 mg of the target compound. ¹H-NMR(300 MHz, CDCl₃-d) δ 1.24-1.27 (m, 4H); δ 1.71-1.74 (m, 2H); δ 2.50 (s,3H); δ 2.63 (s, 3H); δ 2.90 (t, 2H); δ 3.57-3.61 (m, 4H); δ 4.11-4.12(m, 2H); δ 5.30 (m, 2H); δ 6.21-6.22 (d, 1H); δ 7.37-7.39 (d, 1H); δ8.01-8.03 (m, 1H). MS (TOF) 560.5 (M+).

EXAMPLE 25 (1-acetylpiperidin-3-yl)N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate

The methods of Example 8 and Example 11 were respectively carried out,with the use of the product obtained in Example 7 as starting material,the use of t-butyl 3-hydroxy-1-piperidinyl formate in place of t-butyl4-hydroxy-1-piperidinyl formate in Example 8 as starting material, andthe use of acetyl chloride in place of 2,4-dichlorobenzenesulfonylchloride in Example 11, to obtain 150 mg of the target compound. ¹H-NMR(300 MHz, CDCl₃-d) δ 1.72-1.86 (m, 8H); δ 2.07-2.11 (m, 4H); δ 2.73 (s,3H); δ 2.90-2.93 (m, 2H); δ 3.24 (t, 1H); δ 3.53-3.56 (m, 2H); δ 3.93(s, 4H); δ 4.45-4.48 (d, 2H); δ 4.78 (m, 1H); δ 5.85 (s, 1H); δ 8.33 (s,1H). MS (TOF) 391.5 (M+).

EXAMPLE 26[1-(2,2,2-trifluoroacetyl)piperidin-3-yl]N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate

The methods of Example 8 and Example 11 were respectively carried out,with the use of the product obtained in Example 7 as starting material,the use of t-butyl 3-hydroxy-1-piperidinyl formate in place of t-butyl4-hydroxy-1-piperidinyl formate in Example 8 as starting material, andthe use of 3,3,3-trifluoroacetyl chloride in place of2,4-dichlorobenzenesulfonyl chloride in Example 11, to obtain 156 mg ofthe target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.72-1.88 (m, 8H); δ2.78 (s, 3H); δ 2.78-3.14 (m, 4H); δ 3.93 (s, 5H); δ 4.20-4.47 (m, 3H);δ 4.93 (m, 1H); δ 5.83 (s, 1H); δ 8.33 (s, 1H). MS (TOF) 445.4 (M+).

EXAMPLE 27[1-(imidazole-1-carbonyl)piperidin-3-yl]N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate

The methods of Example 8 and Example 11 were respectively carried out,with the use of the product obtained in Example 7 as starting material,the use of t-butyl 3-hydroxy-1-piperidinyl formate in place of t-butyl4-hydroxy-1-piperidinyl formate in Example 8 as starting material, andthe use of CDI in place of 2,4-dichlorobenzenesulfonyl chloride inExample 11, to obtain 153 mg of the target compound. ¹H-NMR (300 MHz,CDCl₃-d) δ 1.73 (m, 5H); 1.95-2.10 (m, 3H); δ 2.75 (s, 3H); δ 2.90 (m,2H); δ 3.37-3.39 (s, 1H); δ 3.62 (d, 1H); δ 3.84-3.85 (d, 2H); δ 3.92(s, 3H); δ 4.26 (m, 1H); δ 4.74 (m, 2H); δ 4.91 (s, 1H); δ 5.87 (s, 1H);δ 7.11 (s, 1H); δ 7.22 (s, 1H); δ 7.89 (s, 1H); δ 8.34 (s, 1H). MS (TOF)443.5 (M+).

EXAMPLE 28[1-(2-thienylsulfonyl)piperidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate

The methods of Example 8 and Example 11 were respectively carried out,with the use of the product obtained in Example 5 as starting material,and the use of 2-thienylsulfonyl chloride in place of2,4-dichlorobenzenesulfonyl chloride in Example 11, to obtain 155 mg ofthe target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.26 (m, 2H); δ1.68-1.91 (m, 9H); δ 2.78 (s, 4H); δ 2.96-3.17 (m, 3H); δ 3.94 (s, 3H);δ 4.12-4.53 (m, 3H); δ 6.21-6.22 (d, 1H); δ 7.13-7.14 (m, 1H); δ7.53-7.54 (m, 1H); δ 7.60-7.61 (d, 1H); δ 8.03-8.04 (m, 1H). MS (TOF)495.6 (M+).

EXAMPLE 291-(1-thienylsulfonyl)piperidin-4-yl]N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate

The methods of Example 8 and Example 11 were respectively carried out,with the use of the product obtained in Example 4 as starting material,and the use of 2-thienylsulfonyl chloride in place of2,4-dichlorobenzenesulfonyl chloride in Example 11, to obtain 150 mg ofthe target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.73 (m, 2H); 1.95-2.10(m, 2H); δ 2.19 (s, 5H); δ 2.65 (m, 3H); δ 2.70-3.39 (m, 7H); δ 3.90 (s,3H); δ 4.84 (m, 3H); δ 4.74 (m, 2H); δ 5.97 (dd, 1H); δ 7.17 (m, 1H); δ7.57 (t, 1H); δ 7.63 (t, 1H); δ 8.10 (dd, 1H). MS (TOF) 495.6 (M+).

EXAMPLE 30N-methyl-N-[1-(4-chloropyrimidin-2-yl)piperidin-4-yl]-[2-(imidazol-1-yl)]acetamide

10 g (1 eq) of the compound obtained in Example 1 was added to a 250 mlthree-necked round-bottom flask, to which trifluoroacetic acid was addedfor the removal of Boc, followed by the addition of potassium carbonateto obtain a free amine. The resulting free amine was dissolved indichloromethane, to which 6.8 g (1.5 eq) of triethylamine was added, andthen 6 g (1.2 eq) of chloroacetyl chloride was added dropwise slowlywithin 30 min in ice-water bath. The reaction was completed after 1 h,and the reaction solution was extracted with dichloromethane and watertwice. The organic phases were combined, dried and concentrated toobtain a crude product. 400 mg of the crude product was subject tofurther reaction by adding in sequence 5 ml DMF, 500 mg K₂CO₃, and 200mg imidazole. The reaction was completed after 4 h, and the reactionsolution was added dropwise slowly to water, to precipitate out 100 mgof the target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.63-1.71 (m, 2H);1.74-1.77 (m, 2H); δ 2.87-2.99 (m, 5H); δ 4.75 (m, 1H); δ 4.85-4.99 (m,4H); δ 6.25-6.53 (dd, 1H); δ 7.02 (s, 1H); δ 7.15 (s, 1H); δ 7.71 (s,1H); δ 8.16-8.17 (dd, 1H). MS (TOF) 334.8 (M+).

EXAMPLE 31N-methyl-N-[1-(4-chloropyrimidin-2-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)acetamide

The method of Example 30 was carried out, with the use of1H-1,2,4-triazole in place of imidazole, to obtain 110 mg of the targetcompound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.63-1.76 (m, 4H); δ 2.85-2.97 (m,5H); δ 4.72 (m, 1H); δ 4.88-4.92 (m, 4H); δ 5.06 (s, 2H); δ 6.51-6.52(dd, 1H); δ 7.98 (s, 1H); δ 8.15-8.16 (dd, 1H); δ 8.26 (s, 1H). MS (TOF)335.8 (M+).

EXAMPLE 32N-methyl-N-[1-(2-chloropyrimidin-4-yl)piperidin-4-yl]-2-(2-chlorobenzoimidazol-1-yl)acetamide

The method of Example 30 was carried out, with the use of the compoundobtained in Example 2 as starting material, and the use of2-chloro-benzoimidazole in place of imidazole, to obtain 111 mg of thetarget compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.66-1.70 (m, 2H); δ1.79-1.82 (m, 2H); δ 2.86-3.00 (m, 5H); δ 4.54 (s, 2H); δ 4.73 (m, 1H);δ 4.97 (s, 2H); δ 6.42-6.44 (d, 1H); δ 7.21-7.22 (m, 1H); δ 7.28-7.31(dd, 1H); δ 7.72-7.40 (dd, 1H), δ 8.05-8.06 (d, 1H). MS (TOF) 419.3(M+).

EXAMPLE 33N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-[2-(imidazol-1-yl)]acetamide

10 g (1 eq) of the compound obtained in Example 7 was added to a 250 mlthree-necked round-bottom flask, to which 6.8 g (1.5 eq) oftriethylamine was added, and then 6 g (1.2 eq) of chloroacetyl chloridewas added dropwise slowly within 30 min in ice-water bath. The reactionwas completed after 1 h, and the reaction solution was extracted withdichloromethane and water twice; the organic phases were combined, driedand concentrated to obtain a crude product; 400 mg of the crude productwas subject to further reaction by adding in sequence 5 ml DMF, 500 mgK₂CO₃, and 200 mg imidazole. The reaction was completed after 4 h, andthe reaction solution was added dropwise slowly to water, to precipitateout 100 mg of the target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.63-1.75(m, 4H); δ 2.85-2.97 (m, 5H); δ 3.92 (s, 3H); δ 4.45-4.49 (m, 2H); δ4.79 (s, 2H); δ 6.98 (s, 1H); δ 7.12 (s, 1H); δ 7.54 (s, 1H); δ 8.33 (s,1H). MS (TOF) 330.4 (M+).

EXAMPLE 34N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)acetamide

The method of Example 33 was carried out, with the use of1H-1,2,4-triazole in place of imidazole, to obtain 113 mg of the targetcompound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.63-1.74 (m, 4H); δ 2.84-2.95 (m,5H); δ 3.91 (s, 3H); δ 4.44-4.48 (m, 2H); δ 4.72 (m, 2H); 5.05 (s, 2H);δ 5.84 (s, 1H); δ 7.97 (s, 1H); δ 8.25 (s, 1H); δ 8.32 (s, 1H). MS (TOF)331.4 (M+).

EXAMPLE 35N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-2-(2-chlorobenzoimidazol-1-yl)acetamide

The method of Example 33 was carried out, with the use of2-chloro-benzoimidazole in place of imidazole, to obtain 101 mg of thetarget compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.68-1.72 (m, 4H); δ2.85-2.93 (dd, 4H); δ 2.97 (s, 3H); δ 3.93 (s, 3H); δ 4.45-4.48 (m, 2H);δ 4.72 (m, 1H); δ 4.94 (s, 2H); δ 5.85 (s, 1H); δ 7.20-7.21 (dd, 1H); δ7.28-7.30 (d, 1H); δ 7.71-7.73 (dd, 1H), δ 8.34 (s, 1H). MS (TOF) 414.9(M+).

EXAMPLE 36N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]-2-{[3-(1,2,4-triazol-1-methyl)]pyrrolidin-1-yl}acetamide

The method of Example 33 was carried out, with the use of the compoundobtained in Example 6 as starting material, and the use ofpyrrolidine-3-methyl-1H-1,2,4-triazole in place of imidazole, to obtain150 mg of the target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.43-1.68 (m,7H); δ 2.00-2.10 (m, 1H); δ 2.51 (s, 3H); δ 2.74 (m, 1H); δ 2.77 (s,3H); δ 2.85-2.97 (m, 2H); δ 3.17-3.60 (m, 3H); δ 3.32 (m, 2H); δ4.14-4.15 (m, 2H); δ 4.54 (s, 2H); δ 4.74 (m, 1H); δ 6.21-6.22 (d, 1H);δ 7.93 (s, 1H); δ 8.02-8.04 (d, 1H), δ 8.20 (s, 1H). MS (TOF) 430.6(M+).

EXAMPLE 37N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]-2-{[2-(1H-1,2,4-triazol-1-methyl)]pyrrolidin-1-yl}acetamide

The method of Example 33 was carried out, with the use of the compoundobtained in Example 5 as starting material, and the use ofpyrrolidine-3-methyl-1H-1,2,4-triazole in place of imidazole, to obtain110 mg of the target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.62-1.66 (m,7H); δ 1.69-1.72 (m, 1H); δ 2.73 (s, 3H); δ 2.78 (m, 1H); δ 2.97 (m,2H); δ 3.17-3.20 (d, 2H); δ 3.34-3.38 (d, 2H); δ 3.94 (s, 3H); δ4.15-4.15 (m, 2H); δ 4.54 (s, 2H); δ 4.74 (m, 1H); δ 6.20-6.22 (d, 1H);δ 7.93 (s, 1H); δ 8.04-8.05 (d, 1H), δ 8.21 (s, 1H). MS (TOF) 414.5(M+).

EXAMPLE 38N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-2-{[2-(1H-1,2,4-triazol-1-methyl)]pyrrolidin-1-yl}acetamide

The method of Example 33 was carried out, with the use of the compoundobtained in Example 7 as starting material, and the use ofpyrrolidine-3-methyl-1H-1,2,4-triazole in place of imidazole, to obtain110 mg of the target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.61-1.67 (m,7H); δ 1.67-1.70 (m, 1H); δ 2.73 (s, 3H); δ 2.77 (m, 1H); δ 2.94 (m,2H); δ 3.16-3.20 (d, 2H); δ 3.29 (d, 2H); δ 3.92 (s, 3H); δ 4.13-4.15(m, 2H); δ 4.43-4.47 (d, 2H); δ 4.72 (m, 1H); δ 5.84 (s, 1H); δ 7.92 (s,1H); δ 8.20 (s, 1H), δ 8.32 (s, 1H). MS (TOF) 414.5 (M+).

EXAMPLE 39N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]-2-{[2-(1H-1,2,4-triazol-1-methyl)]pyrrolidin-1-yl}acetamide

The method of Example 33 was carried out, with the use of the compoundobtained in Example 4 as starting material, and the use ofpyrrolidine-3-methyl-1H-1,2,4-triazole in place of imidazole, to obtain109 mg of the target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.59-1.65 (m,6H); δ 1.66-1.69 (m, 1H); δ 2.04 (m, 1H); δ 2.76 (m, 1H); δ 2.79 (s,3H); δ 2.93 (m, 2H); δ 3.19-3.22 (m, 2H); δ 3.35-3.39 (d, 2H); δ 3.90(s, 3H); δ 4.16 (m, 2H); δ 4.88-4.92 (m, 1H); δ 5.31 (m, 1H); δ5.99-6.00 (d, 1H); δ 7.93 (s, 1H); δ 8.04-8.06 (d, 1H), δ 8.21 (s, 1H).MS (TOF) 414.5 (M+).

EXAMPLE 40N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)acetamide

The method of Example 33 was carried out, with the use of the compoundobtained in Example 7 as starting material, and the use of1,2,4-triazole in place of imidazole, to obtain 115 mg of the targetcompound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.37-1.40 (m, 3H); δ 1.98-2.02 (m,2H); δ 3.06-3, 07 (m, 2H); δ 3.93 (s, 3H); δ 4.07-4.09 (m, 1H); δ4.23-4.26 (m, 2H); δ 4.87 (s, 2H); δ 5.84 (s, 1H); δ 6.40 (s, 1H); δ8.06 (s, 1H); δ 8.20 (s, 1H); δ 8.34 (s, 1H). MS (TOF) 400.9 (M+).

EXAMPLE 41N-methyl-N-[1-(6-methylpyrimidin-4-yl)piperidin-4-yl]-2-(2-chloro-benzoimidazol-1-yl)acetamide

The methods of Example 1 and Example 33 were respectively carried out,with the use of 2-chloro-4-methylpyrimidine as starting material, andthe use of 2-chloro-benzoimidazole in place of imidazole in Example 33,to obtain 105 mg of the target compound. ¹H-NMR (300 MHz, CDCl₃-d) δ1.70-1.73 (m, 4H); δ 2.35 (s, 3H); δ 2.86-2.93 (m, 3H); δ 2.98 (s, 2H);δ 4.95 (m, 1H); δ 4.98 (s, 3H); δ 5.06 (m, 1H); δ 6.39-6.40 (d, 1H); δ7.23-7.21 (dd, 1H); δ 7.29-7.30 (m, 2H); δ 7.71-7.74 (t, 1H), δ8.17-8.20 (d, 1H). MS (TOF) 398.9 (M+).

EXAMPLE 42N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)acetamide

The method of Example 33 was carried out, with the use of the compoundobtained in Example 4 as starting material, and the use of1,2,4-triazole in place of imidazole, to obtain 120 mg of the targetcompound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.65-1.73 (m, 4H); δ 2.84-2.89 (m,2H); δ 2.92 (s, 3H); δ 3.88 (s, 3H); δ 4.60-4.71 (m, 1H); δ 4.87-4.90(m, 2H); δ 5.05 (s, 2H); δ 5.96-5.89 (d, 1H); δ 7.91 (s, 1H); δ8.02-8.04 (d, 1H), δ 8.26 (s, 1H). MS (TOF) 331.4 (M+).

EXAMPLE 43N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)acetamide

The method of Example 33 was carried out, with the use of the compoundobtained in Example 5 as starting material, and the use of1,2,4-triazole in place of imidazole, to obtain 150 mg of the targetcompound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.63-1.77 (m, 4H); δ 2.94 (s, 3H);δ 2.96-2.99 (m, 2H); δ 3.94-3.95 (s, 3H); δ 4.56 (m, 1H); δ 4.74 (m,2H); δ 5.07 (s, 2H); δ 6.21-6.22 (d, 1H); δ 7.98 (s, 1H); δ 8.04-8.06(d, 1H), δ 8.26 (s, 1H). MS (TOF) 331.4 (M+).

EXAMPLE 44N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)acetamide

The method of Example 33 was carried out, with the use of the compoundobtained in Example 6 as starting material, and the use of1,2,4-triazole in place of imidazole, to obtain 125 mg of the targetcompound. ¹H-NMR (300 MHz, CDCl₃-d) δ 1.63-1.77 (m, 4H); δ 2.50 (s, 3H);δ 2.83-2.93 (m, 5H); δ 4.53-4.56 (m, 2H); δ 4.72 (m, 1H); δ 5.06 (s,2H); δ 6.21-6.22 (d, 1H); δ 7.98 (s, 1H); δ 8.02-8.03 (d, 1H), δ 8.24(s, 1H). MS (TOF) 347.4 (M+).

EXAMPLE 45

Antitumor bioactivity evaluation of compounds of the present invention

1. Test Materials

Cells:

Name Generation Survival rate % BT474 P103 100 BT/Lap^(R)1.0 / 97.12MDA-MB-361 F72 97.76 SK-BR3 F28 95.95 SK/Lap^(R)1.0 / 93.35 MDA-MB-453F8 96.8 Note: BT474, SK-BR3 are human breast cancer cell lines sensitiveto lapatinib, BT/LapR, SK/LapR are human breast cancer cell lines withsecondary resistance to lapatinib after being stimulated with lapatinib,MDA-MB-361 and MDA-MB-453 are human breast cancer cell lines withnatural resistance to lapatinib. BT474, MDA-MB-361, SK-BR3 andMDA-MB-453 all come from American Type Culture Collection (ATCC).

Lapatinib: 10 Mm in DMSO, BioVision, Cat: 1624-100, Lot: 50324;

ATPlit kit: CellTiter-Glo Substrate, Promega, Part: G755B, Lot:32513501, EXP: 2014-05.

2. Experimental Procedures

Cells plating: a 100 mm culture dish with overgrown adherent cells wasdigested with 1 ml 0.25% trypsin (GIBCO) at 37° C. for 5 min, and thereaction was terminated with 2 ml culture medium (containing 10% FBS,GIBCO). The cells were scattered and collected, after counting, dilutedto 1×10⁵ cells/ml, and seeded into a 96-well plate at 50 μl/well, 5000cells/well, excluding the peripheral circle of wells to which no cellwas added, but PBS was added, 60 wells in total, and then incubated at37° C. for 24 hours for adhering.

Addition of compound and lapatinib: the test sample was diluted to afinal concentration of 5 μM, culture medium with the correspondingcompound solvent concentration was filled in control well, the compoundsolvent concentration was kept consistent in each well, 5 parellel wellswere set for each concentration, 25 μl each well, 25 μl culture medium(in combined group, 25 μl lapatinib was added till a final concentrationof 1 μM) supplemented, followed by incubation at 37° C. for 72 hours.

Test after incubation at 37° C. for 72 hours: 50 μL of ATPlite kitsubstrate solution was added in each well, and vibrated for 3 min,followed by placing aside in darkness for 10 min, and then supernatant100 μl/well was taken and placed on aluminescence test plate; theluminescence test plate that has been fully incubated was placed in aluminescence test instrument, to read luminescence value.

3. Data Processing

(1) Cell survival rate (%)=Experimental group RLU/control groupRLU×100%. The experimental data were subjected to data analysis andprocessing by utilizing GraphPad software. The results were shown inTable 1.

(2) The nature of interaction between two drugs was evaluated by usingcoefficient of drug interaction (CDI), CDI being calculated as follows:CDI=AB/(A×B)×100%. As calculated according to the number of viable cells(luminescencevalue), when CDI<1, the nature of interaction between twodrugs is synergism; when CDI<0.7, the synergism is very significant;when CDI=1, the nature of interaction between two drugs is addition;when CDI>1, the nature of interaction between two drugs is antagonism.

The coefficient of drug interaction (CDI) between the compound ofExample 30 and Lapatinib was shown in Table 1: CDI was less than 1 inboth breast cancer MDA-MB-361 and SK/Lap^(R)1.0 cell lines, inparticular CDI<0.7 in SK/Lap^(R)1.0, which indicated that the synegerismbetween the compound of Example 30 and Lapatinib was very significant.

TABLE 1 Example Survival Cell 30 (μM) RLU Average rate % BT474 101507553 1590600 1557681 1648956 147837 1556633 93.5 0 1622024 16594301732751 1740236 156591 1664070 100.0 BT/Lap^(R) 10 1032464 11170091137209 1139456 110653 1106535 92.5 1.0 0 1137956 1191829 12307311194819 122773 1196615 100.0 MDA- 10 420320 423604 472624 461509 459483447508 104.1 MB-361 0 402997 429160 446144 429977 441034 429862 100.0SK-BR3 10 1937000 2054461 2105337 2128527 211132 2067329 83.8 0 22759192477176 2507850 2556480 252281 2468047 100.0 SK/Lap^(R) 10 9344591005533 1044437 1098307 105641 1051172 94.2 1.0 0 858893 984583 10751111181353 122175 1115700 100.0 MDA- 10 2644764 2710604 2763721 2789907277195 2736190 82.8 MB-453 0 2894653 3278459 3246290 3306141 3388433304832 100.0 Lapatinib 0 Example Survival Cell 30 (μM) RLU Average rate% CDI BT474 10 398377 392813 409887 415611 444171 412172 24.8 1.05 0412434 392000 415243 442519 426263 417692 25.1 BT/Lap^(R) 10 598830589681 617719 609377 608990 604919 50.6 1.03 1.0 0 620030 626799 621089658350 647589 634771 53.0 MDA- 10 288984 301731 291331 297614 306466297225 69.1 0.94 MB-361 0 302584 295553 303233 301683 309791 302569 70.4SK-BR3 10 610944 595881 594551 631641 634386 613481 24.9 1.13 0 620929634466 661000 652027 675276 648740 26.3 SK/Lap^(R) 10 609037 638566657327 858149 886574 634977 56.9 0.65 1.0 0 633107 910516 1026481 1053411147684 103452 92.7 MDA- 10 1215769 1250183 105341 130180 123372 12109836.6 1.26 MB-453 0 1036211 1128233 113422 133323 1171624 116070 35.1Lapatinib 1

EXAMPLE 46 Measurement of Affinity Between Compounds of Examples andHsp70

Materials and Method

1. Instrument: BIACORE T100 biomolecular interaction analyzer (GE, USA)

2. Reagents: PBS Buffer (×10), P20, CM5 chip (GE, USA), Hsp70 (human,ADI-ESP-550-D), manufactured by Enzo Life Sciences.

Formulation of Compound

30 mM mother liquor of compound was formulated with DMSO, which wasdiluted to 2 mM application solution with DMSO before use. 5 μl of theapplication solution was taken and diluted with 95 μl 1.05×PBS to 100μM, and then diluted in turn with PBS containing 5% DMSO to 10, 3, 1,0.3, 0.1, 0.03, 0.01, 0.003 μM, 0.3 μM. PBS containing 5% DMSO was usedas solvent control.

Operating Procedures

1. Coupling of Hsp70 Protein and CM5 Chip

Hsp70 protein was diluted with 10 mmol/L sodium acetate buffer solution(pH 5.0) to 30 μg/ml, and coupled directly on a hydrophiliccarboxylmethyl dextran matrix sensor chip M5 through normalized primaryamine coupling reaction, RU=11209, and then the chip was balanced for1-2h with PBS Buffer at constant current.

2. Measurement of Affinity Between Compound and Hsp70 Protein

At a flow rate of 30 μl/ml, with PBS containing 5% DMSO as mobile phase,at 25° C., the compound was introduced in the sequence of 0.003, 0.01,0.03, 0.1, 0.3, 1, 3 and 10 with binding time 90 seconds, anddissociation time 120 seconds.

3. Result Analysis:

According to binding characteristics between the compounds and protein,the binding constant (equilibrium dissociation constant KD) betweendrugs and proteinwas calculated by selecting steady state model with theformula: Conc*Rmax/[conc+KD]+offset.

4. The results of preliminary screeing showed that, among the compoundsof Examples as screened, the compounds of Example 1, Example 2, Example3, Example 5, Example 6, Example 13, Example 30 and Example 42, at 10μm, produced an effect equivalent to that of positive compoundVER-155008 (as reported in the literature, it was a compound having adefinite binding to Hsp70, Cancer Chemother Pharmacol 2010, 66:535-545). This indicated that the affinity for Hsp70 of the compounds ofthe above Examples was equivalent to that of the positive compoundVER-155008 (literature value 0.3 μM).

5. Measurement of affinity of compounds for HSP70 protein (KD value)

At a flow rate of 30 μl/ml, PBS containing 5% DMSO as mobile phase, at25° C., the compound was injected in the order of 0.003, 0.01, 0.03,0.1, 0.3, 1, 3, and 10 μM, with the binding time of 90 sec, and thedissociation time of 120 sec. The affinities of 44 screened compoundsfor HSP70 protein were measured (the results were shown in Table 2).

TABLE 2 Name of compound KD (mol/L) VER-155008  3.00 × 10⁻⁷ Example 12.191 × 10⁻⁶ Example 2 2.466 × 10⁻⁷ Example 3 5.248 × 10⁻⁷ Example 61.272 × 10⁻⁵ Example 13 5.199 × 10⁻⁷ Example 30 2.249 × 10⁻⁷ Example 424.339 × 10⁻⁷

Although specific embodiments of the present invention have beendescribed in detail, a person skilled in the art will appreciate that,according to all the teachings that have been disclosed, variousmodifications and substitutions can be conducted to the details, and allof these changes are within the protection scope of the presentinvention. The entire scope of the present invention is given by theappended claims and any equivalents thereof.

What is claimed is:
 1. A compound of formula (I), an isomer, apharmaceutically acceptable salt or a solvate thereof,

wherein: A represents CH₂, S, O,

wherein “

” terminal is attached to R₂; R₁ represents aryl, aromatic heterocyclyl,arylalkyl, or aromatic heterocyclylalkyl, wherein said aryl, aromaticheterocyclyl, arylalkyl, or aromatic heterocyclylalkyl is unsubstitutedor substituted with one or two substituents independently selected fromthe group consisting of halogen, nitro, hydroxy, amino, cyano, alkyl,alkoxy, alkylthio, alkylamino, cycloalkoxy, cycloalkylthio,cycloalkylamino, alkenyl and alkynyl; R₂ represents hydrogen, alkyl,cycloalkyl, substituted cycloalkyl, alkoxy, alkoxycarbonyl, alkanoyl,substituted alkanoyl, aliphatic heterocyclyl, substituted aliphaticheterocyclyl, aliphatic heterocyclylalkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, arylsulfonyl, substitutedarylsulfonyl, arolyl, substituted arolyl, aromatic heterocyclyl,substituted aromatic heterocyclyl, aromatic heterocyclylalkyl, aromaticheterocyclylsulfonyl, aromatic heterocyclylacyl; wherein the substituentincludes alkyl, halogen, nitro, cyano, amino, hydroxy, alkoxy,mono(C₁-C₆)alkylamino, di(C₁-C₆)alkylamino, C₂-C₆alkenyl, C₂-C₆alkynyl,C₁-C₆haloalkyl, C₁-C₆haloalkoxy, aromatic heterocyclylalkyl; R₃represents hydrogen, C₁-C₃alkyl, or C₃-C₆cycloalkyl.
 2. The compound offormula (I), an isomer, a pharmaceutically acceptable salt or a solvatethereof according to claim 1, wherein: R₁ represents aromaticheterocyclyl or substituted aromatic heterocyclyl, wherein thesubstituent is selected from the group consisting of halogen, nitro,amino, cyano, C₁-C₆alkyl, C₁-C₆alkoxy, and C₁-C₆alkylthio.
 3. Thecompound of formula (I), an isomer, a pharmaceutically acceptable saltor a solvate thereof according to claim 2, wherein: R₁ representspyrimidinyl or thiazolyl, said pyrimidinyl or thiazolyl beingunsubstituted or substituted with one or two substituents independentlyselected from the group consisting of methyl, halogen, nitro, amino,cyano, methoxy, ethoxy, methylthio and ethylthio.
 4. The compound offormula (I), an isomer, a pharmaceutically acceptable salt or a solvatethereof according to claim 1, wherein: R₂ represents hydrogen,C₁-C₆alkyl, C₁-C₆alkoxycarbonyl, C₁-C₆alkanoyl, substitutedC₁-C₆alkanoyl, 5- or 6-membered aliphatic heterocyclyl, substituted 5-or 6-membered aliphatic heterocyclyl, aryl, substituted aryl,arylC₁-C₆alkyl, substituted arylC₁-C₆alkyl, arylsulfonyl, substitutedarylsulfonyl, aromatic heterocyclyl, substituted aromatic heterocyclyl,aromatic heterocyclylsulfonyl, aromatic heterocyclylacyl, wherein thesubstituent is selected from the group consisting of C₁-C₆alkyl,halogen, nitro, cyano, amino, C₁-C₆alkoxy, aromaticheterocyclylC₁-C₃alkyl.
 5. The compound of formula (I), an isomer, apharmaceutically acceptable salt or a solvate thereof according to claim1, wherein: R₃ represents hydrogen, or methyl.
 6. The compound offormula (I), an isomer, a pharmaceutically acceptable salt or a solvatethereof according to claim 1, wherein: A represents CH₂, O, S,

wherein “

” terminal is attached to R₂; R₁ represents pyrimidinyl, wherein saidpyrimidinyl is unsubstituted or substituted with one or two substituentsindependently selected from the group consisting of methyl, halogen,nitro, amino, cyano, methoxy, ethoxy, methylthio and ethylthio; R₂represents hydrogen, t-butyl, t-butoxycarbonyl, thiazol-2-sulfonyl,imidazol-1-formyl, phenyl, 1-naphthyl, 2-naphthyl, 4-fluorophenyl,imidazol-1-yl, triazol-1-yl, 2-chloro-benzimidazol-1-yl,2,3-dichlorobenzenesulfonyl, 2,4-chlorobenzenesulfonyl, 3-chlorobenzyl,4-chlorobenzyl, 4-fluorobenzyl, acetyl, trifluoroacetyl,1,2,4-triazol-1-methylpyrrolidin-1-yl; R₃ represents hydrogen, ormethyl.
 7. The compound of formula (I), an isomer, a pharmaceuticallyacceptable salt or a solvate thereof according to claim 1, which isselected from the following compounds: (1) t-butylN-methyl-N-[1-(4-chloropyrimidin-2-yl)piperidin-4-yl]carbamate; (2)t-butyl N-methyl-N-[1-(2-chloropyrimidin-4-yl)piperidin-4-yl]carbamate;(3) t-butylN-methylN-[1-(6-chloropyrimidin-4-yl)piperidin-4-yl]carbamate; (4)t-butyl N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate;(5) t-butylN-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate; (6)t-butylN-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate; (7)t-butyl N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate;(8) (1-t-butoxycarbonylpiperidin-4-yl)N-methyl-N-[1-(2-methylthiopyrimidin-4-yl) piperidin-4-yl]carbamate; (9)(S)-(1-t-butoxycarbonylpyrrolidin-3-yl)N-methyl-N-[1-(4-methylthiopyrimidin-2-yl) piperidin-4-yl]carbamate;(10) (S)-(pyrrolidin-3-yl)N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamatehydrochloride; (11)[1-(2,4-dichlorobenzenesulfonyl)piperidin-4-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate;(12)(S)-[1-(imidazole-1-carbonyl)pyrrolidin-3-yl]N-methyl-N-[1-(2-methyloxypyrimidin-4-yl)piperidin-4-yl]carbamate;(13)(S)-[1-(2-thienylsulfonyl)pyrrolidin-3-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate; (14) p-fluorophenylthioN-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate; (15)p-fluorophenylthioN-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate; (16)(1-acetylpiperidin-4-yl)N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate; (17)(S)-[1-(2-thienylsulfonyl)pyrrolidin-3-yl]N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate; (18)[1-(3-chlorobenzyl)piperidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate; (19)[1-(3-chlorobenzyl)piperidin-4-yl]N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate; (20)[1-(2,3-dichlorobenzenesulfonyl)piperidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate;(21)[1-(2,4-dichlorobenzenesulfonyl)piperidin-4-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate;(22)[1-(4-fluorobenzyl)piperidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate; (23)(S)-[1-(2,3-dichlorobenzenesulfonyl)pyrrolidin-3-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate;(24)(S)-[1-(2,4-dichlorobenzenesulfonyl)pyrrolidin-3-yl]N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]carbamate;(25) (1-acetylpiperidin-3-yl)N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate; (26)[1-(2,2,2-trifluoroacetyl)piperidin-3-yl]N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate; (27)[1-(imidazole-1-carbonyl)piperidin-3-yl]N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate; (28)[1-(2-thienylsulfonyl)piperidin-3-yl]N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]carbamate; (29)[1-(1-thienylsulfonyl)piperidin-4-yl]N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]carbamate; (30)N-methyl-N-[1-(4-chloropyrimidin-2-yl)piperidin-4-yl]-[2-(imidazol-1-yl)]acetamide;(31)N-methyl-N-[1-(4-chloropyrimidin-2-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)acetamide; (32)N-methyl-N-[1-(2-chloropyrimidin-4-yl)piperidin-4-yl]-2-(2-chloro-benzoimidazol-1-yl)acetamide;(33)N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-[2-(imidazol-1-yl)]acetamide;(34)N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)acetamide; (35)N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-2-(2-chloro-benzoimidazol-1-yl)acetamide;(36)N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]-2-{[3-(1H-1,2,4-triazol-1-methyl)]pyrrolidin-1-yl}acetamide;(37)N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]-2-{[2-(1H-1,2,4-triazol-1-methyl)]pyrrolidin-1-yl}acetamide;(38)N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-2-{[2-(1H-1,2,4-triazol-1-methyl)]pyrrolidin-1-yl}acetamide;(39)N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]-2-{[2-(1H-1,2,4-triazol-1-methyl)]pyrrolidin-1-yl}acetamide;(40)N-methyl-N-[1-(6-methoxypyrimidin-4-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)acetamide; (41)N-methyl-N-[1-(6-methylpyrimidin-4-yl)piperidin-4-yl]-2-(2-chloro-benzoimidazol-1-yl)acetamide; (42)N-methyl-N-[1-(4-methoxypyrimidin-2-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)acetamide; (43)N-methyl-N-[1-(2-methoxypyrimidin-4-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)acetamide; (44)N-methyl-N-[1-(2-methylthiopyrimidin-4-yl)piperidin-4-yl]-2-(1H-1,2,4-triazol-1-yl)acetamide.
 8. A method for preparing the compound of formula (I), anisomer, a pharmaceutically acceptable salt or a solvate thereofaccording to claim 1, which comprises the following steps: (1) compound2 and compound 1 undergo nucleophilic substitution reaction to obtaincompound 3; compound 3 is attacked by nucleophilic agent Nu⁻ to obtaincompound 4; and compound 4 undergoes amino deprotection and amidationreaction respectively to obtain an active intermediate 6; wherein, incompound 1, A, B, C represent C or N atom, and at least one of them is Catom; L is a conventional leaving group such as halogen, two Ls may bethe same or different; Pg is an amino protective group such as Boc; R₁and R₃ are as defined in claim 1; compound 6 is an amidation product ofcompound 5, according to different amidation reagent, compound 6 may berespectively the following three forms 6-1, 6-2 or 6-3;

(2) according to different definition of A, the compound of formula (I)as claimed in claim 1 can be prepared by the following stepsrespectively: when A is CH₂, R₂H is nucleophilic agent, such as amine;R₂H and compound 6-1 directly undergo nucleophilic substitution reactionto obtain compound I-1; wherein R₁, R₂, R₃ are as defined in claim 1:

when A is S, R₂SH is nucleophilic agent, such as thiophenol; R₂SH andcompound 6-2 directly undergo nucleophilic substitution reaction toobtain compound I-2; wherein R₁, R₂, R₃ are as defined in claim 1; L isa conventional leaving group, such as halogen;

when A is

 m=1, 2, n=2, 3; and when m=1, n=2 or 3; and when m=2, n=2; compound 6-3and compound 9 undergo nucleophilic addition reaction to obtain compound7; compound 7 undergoes amino deprotection to obtain compound 8;finally, compound 8 and R₂L undergo nucleophilic substitution reactionto obtain compound I-3; wherein R₁, R₂, R₃ are as defined in claim 1; Pgis a conventional amino protective group, such as Boc;


9. The method according to claim 8, which, when R₁ is a pyrimidine ring,comprises the following reaction route, i.e., first,2,4-dichloro-pyrimidine or 4,6-dichloro-pyrimidine as starting materialreacts with t-butyl N-methyl-N-(piperidin-4-yl)carbamate in a solventsuch as THF or DMF, at normal temperature, in the presence of potassiumcarbonate, sodium carbonate or sodium bicarbonate, to obtain compound3A; compound 3A then reacts with sodium methoxide, sodium ethoxide orsodium thiomethoxide in the corresponding alcohol or THF solvent underrefluxing conditions to obtain compound 4A; compound 4A undergoes BOCremoval under the action of trifluoroacetic acid in DCM solvent at roomtemperature to obtain intermediate 5A; intermediate 5A reacts with CDIin DMF under heating conditions to obtain compound 6A; compound 6A andthe corresponding alcohol dissolved in DMF or DMSO react with compounds7A-7D respectively at room temperature under the catalysis of NaH toobtain compounds 8A-8D; compounds 8B, 8C, 8D respectively undergodeprotection under the action of trifluoroacetic acid in DCM at roomtemperature to obtain compounds 9B, 9C, 9D; compounds 9B, 9C, 9Drespectively undergo condensation reaction with the correspondingsulfonyl chloride 11 (1) or acid chloride 11 (2) in a solvent such asTHF or DMF at room temperature, while neutralizing acid generated duringthe reaction using potassium carbonate, sodium carbonate, sodiumbicarbonate, to obtain compounds (1)B-10(1)D and 10(2)B-10(2)D of thepresent invention; or intermediate 5A dissolved in DCM undergoescondensation reaction with 2-chloroacetyl chloride in an ice bath, whileneutralizing acid generated during the reaction using TEA and the like,to obtain compound 12A; compound 12A then reacts with the correspondingamine in a solvent such as DMF or THF at room temperature, in thepresence of potassium carbonate, sodium carbonate or sodium bicarbonate,to obtain compound 13A of the present invention; then, separating andpurifying the product,

wherein, compounds 11 (1) and 11 (2) mentioned in the above formula arerespectively:

 7A-7D, 8B-8D, 9B-9D, 10 (1)B-10 (1)D and 10 (2)B-10 (2)D arerespectively: A B C  7 R₇—SH

 8

 9

10(1)

10(2)

D  7

 8

 9

10(1)

10(2)


10. A pharmaceutical composition, comprising the compound of formula(I), an isomer, a pharmaceutically acceptable salt or a solvate thereofaccording to claim 1, and a pharmaceutically acceptable carrier orexcipient.
 11. The pharmaceutical composition according to claim 10,which further comprises one or more of other antitumor drugs forexample. 12.-15. (canceled)
 16. A method for preventing and/or treatingdrug-resistant tumors or diseases or conditions caused by drug-resistantbacteria, which comprises administering to a subject in need of suchprevention or treatment a prophylactically and/or therapeuticallyeffective amount of the compound of formula (I), an isomer, apharmaceutically acceptable salt or a solvate thereof according toclaim
 1. 17. A method for preventing and/or treating tumors,neurodegenerative diseases, allograft rejection, and diseases orconditions related to infection which comprises administering to asubject in need of such prevention or treatment a prophylacticallyand/or therapeutically effective amount of the compound of formula (I),an isomer, a pharmaceutically acceptable salt or a solvate thereofaccording to claim
 1. 18. The method according to claim 17, wherein thetumors are cancers selected from the group consisting of breast cancer,prostate cancer, liver cancer, esophageal cancer, gastric cancer, andskin cancer.
 19. The method according to claim 17, wherein theneurodegenerative diseases are selected from the group consisting ofAlzheimer's disease, amyotrophic lateral sclerosis, ataxiatelangiectasia, Creutzfeldt-Jakob disease, Huntington's disease,cerebellar atrophy, multiple sclerosis, Parkinson's disease, primarylateral sclerosis, and spinal muscular atrophy. 20.-23. (canceled)
 24. Amethod for resisting/reversing drug resistance of bacteria or drugresistance of tumor cells in cells, which comprises administering to thecells an effective amount of the compound of formula (I), an isomer, apharmaceutically acceptable salt or a solvate thereof according toclaim
 1. 25. A method for inhibiting expression of Heat shock protein 70(Hsp70) in cells, which comprises administering to the cells aneffective amount of the compound of formula (I), an isomer, apharmaceutically acceptable salt or a solvate thereof according toclaim
 1. 26. The method according to claim 24, wherein the cells arecell lines, or cells from a subject.
 27. The method according to claim24, wherein the tumor cells are cells selected from the group consistingof breast cancer cells, prostate cancer cells, liver cancer cells,esophageal cancer cells, gastric cancer cells, and skin cancer cells.28. The method according to claim 24, wherein the method is carried outin vitro.
 29. The method according to claim 24, wherein the method iscarried out in vivo. 30.-41. (canceled)
 42. A kit forresisting/reversing drug resistance of bacteria or drug resistance oftumor cells in cells, which comprises the compound of formula (I), anisomer, a pharmaceutically acceptable salt or a solvate thereofaccording to claim
 1. 43. A kit for inhibiting activity of Heat shockprotein 70 (Hsp70) in cells, which comprises the compound of formula(I), an isomer, a pharmaceutically acceptable salt or a solvate thereofaccording to claim 1.